Charged ion channel blockers and methods for use

ABSTRACT

The invention provides compounds of Formula (I), or pharmaceutically acceptable salts thereof:The compounds, compositions, methods and kits of the invention are useful for the treatment of pain, cough, itch, and neurogenic inflammation.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/815,803, filed Mar. 11, 2020, which claims the benefit of U.S.Provisional Application Ser. No. 62/816,441 filed Mar. 11, 2019 and U.S.Provisional Application Ser. No. 62/931,599 filed Nov. 6, 2019. Theentire contents of the above applications are incorporated by referenceherein.

BACKGROUND OF THE INVENTION

The invention features compounds, compositions and methods for theselective inhibition of sensory neurons (nociceptors, cough receptorsand pruriceptors) and the treatment of neurogenic inflammation bytargeting nociceptors with a small molecule drug, while minimizingeffects on non-nociceptive neurons or other types of cells. According tothe method of the invention, small, cationic drug molecules gain accessto the intracellular compartment of sensory neurons via entry throughlarge pore receptor/ion channels that are present in pain-cough- anditch-sensing neurons but to a lesser extent or not at all in other typesof neurons or in other types of tissue.

Local anesthetics such as lidocaine and articaine act by inhibitingvoltage-dependent sodium channels in neurons. These anesthetics blocksodium channels and thereby the excitability of all neurons, not justpain-sensing neurons (nociceptors). Thus, while the goal of topical orregional anesthesia is to block transmission of signals in nociceptorsto prevent pain, administration of local anesthetics also producesunwanted or deleterious effects such as general numbness from block oflow threshold pressure and touch receptors, motor deficits and/orparalysis from block of motor axons and other complications from blockof autonomic fibers. Local anesthetics are relatively hydrophobicmolecules that gain access to their blocking site on the sodium channelby diffusing through the cell membrane. Charged derivatives of thesecompounds, which are not membrane-permeable, have no effect on neuronalsodium channels when applied to the external surface of the nervemembrane but can block sodium channels if somehow introduced inside thecell, for example by diffusion from a micropipette used for whole-cellelectrophysiological recording from isolated neurons. Pain-, cough-, anditch-sensing neurons differ from other types of neurons in expressing(in most cases) the TRPV1 receptor/channel, which is activated bypainful heat or by capsaicin, the pungent ingredient in chili pepper.Other types of channels selectively expressed in various types ofpain-sensing, cough-sensing and itch-sensing (pruriceptor) neuronsinclude but are not limited to TRPV2-4, TRPA1, TRPM8, ASIC and P2X(2/3)channels. It is well established that some cationic small molecules suchas QX-314 are able to enter a cell via passage through activated largepore channels such as TRPV1.

Neuropathic, inflammatory, and nociceptive pain differ in theiretiology, pathophysiology, diagnosis, and treatment. Nociceptive painoccurs in response to the activation of a specific subset of highthreshold peripheral sensory neurons, the nociceptors, by intense ornoxious stimuli. It is generally acute, self-limiting and serves aprotective biological function by acting as a warning of potential oron-going tissue damage. It is typically well-localized. Examples ofnociceptive pain include, but are not limited to, traumatic or surgicalpain, labor pain, sprains, bone fractures, burns, bumps, bruises,injections, dental procedures, skin biopsies, and obstructions.

Inflammatory pain is pain that occurs in the presence of tissue damageor inflammation including postoperative (i.e. pain associated with acuteperioperative pain resulting from inflammation caused by tissue trauma(e.g., surgical incision, dissection, burns) or direct nerve injury(e.g., nerve transection, stretching, or compression)), post-traumaticpain, arthritic pain (rheumatoid; or osteoarthritis (i.e. joint pain andstiffness due to gradual deterioration of the joint cartilage; riskfactors include aging, injury, and obesity; commonly affected joints arethe hand, wrist, neck, knee, hip, and spine)), pain and pain associatedwith damage to joints, muscle, and tendons as in axial low back pain(i.e. a prevalent, painful condition affecting the lower portion of theback; common causes include muscle strain, spine fracture, bulging orruptured disc, and arthritis), severe nociceptive pain may transition toinflammatory pain if there is associated tissue injury.

Neuropathic pain is a common type of chronic, non-malignant pain, whichis the result of an injury or malfunction in the peripheral or centralnervous system and serves no protective biological function. It isestimated to affect more than 1.6 million people in the U.S. population.Neuropathic pain has many different etiologies, and may occur, forexample, due to trauma, surgery, herniation of an intervertebral disk,spinal cord injury, diabetes, infection with herpes zoster (shingles),HIV/AIDS, late-stage/cancer, amputation (including mastectomy), carpaltunnel syndrome, chronic alcohol use, exposure to radiation, and as anunintended side-effect of neurotoxic treatment agents, such as certainanti-HIV and chemotherapeutic drugs. Peripheral neuropathy is caused bydamages to the peripheral nerves from injury, trauma, prolongedpressure, or inflammation causing numbness and pain in correspondingareas of the body.

Neuropathic pain is frequently described as “burning,” “electric,”“tingling,” or “shooting” in nature. It is often characterized bychronic dynamic allodynia (defined as pain resulting from a movingstimulus that does not ordinarily elicit a painful response, such aslight touch) and hyperalgesia (defined as an increased sensitivity to anormally painful stimulus), and may persist for months or years beyondthe apparent healing of any damaged tissues.

Pain may occur in patients with cancer, which may be due to multiplecauses; inflammation, compression, invasion, metastatic spread into boneor other tissues.

There are some conditions where pain occurs in the absence of a noxiousstimulus, tissue damage or a lesion to the nervous system, calleddysfunctional pain and these include but are not limited tofibromyalgia, tension type headache, and irritable bowel disorders.

Migraine is a headache associated with the activation of sensory fibersinnervating the meninges of the brain.

Itch (pruritus) is a dermatological condition that may be localized andgeneralized and can be associated with skin lesions (rash, atopiceczema, wheals). Itch accompanies many conditions including but notlimited to stress, anxiety, UV radiation from the sun, metabolic andendocrine disorders (e.g., liver or kidney disease, hyperthyroidism),cancers (e.g., lymphoma), reactions to drugs or food, parasitic andfungal infections, allergic reactions, diseases of the blood (e.g.,polycythemia vera), and dermatological conditions. Itch is mediated by asubset of small diameter primary sensory neurons, the pruriceptor, thatshare many features of nociceptor neurons, including but not limited toexpression of TRPV1 channels, and other large pore channels (e.g.,TRPV2-4, TRPA1, TRPM8, ASIC and P2X(2/3). Certain itch mediators-such aseicosanoids, histamine, bradykinin, ATP, and various neurotrophins haveendovanilloid functions. Topical capsaicin suppresses histamine-induceditch. Pruriceptors like nociceptors are therefore a suitable target forthis method of delivering ion channel blockers.

Cough is a defensive reflex designed to protect the airway from foreignbodies and to aid in the clearance of luminal debris. This reflex,however, can became aberrant in a number of diseases leading to anon-productive dry cough where hyper- or allo-tussive states exist.Hyper- and allo-tussive states are often chronic in nature lastinggreater than three months and can be manifested in many airway diseasesstates including asthma, COPD, asthma-COPD overlap syndrome (ACOS),interstitial pulmonary fibrosis (IPF) and lung cancer. In addition,inappropriate cough reflexes can be manifested acutely and chronicallyfollowing viral infection. Furthermore, chronic cough can be idiopathicin nature with unknown etiology.

Neurogenic inflammation is a mode of inflammation mediated by theefferent (motor) functions of sensory neurons, in which pro-inflammatorymediator molecules released in the periphery by pain-sensing neurons(nociceptors) both activate a variety of inflammatory pathways in immunecells and also act on the vascular system to alter blood flow andcapillary permeability.

Neurogenic inflammation contributes to the peripheral inflammationelicited by tissue injury, autoimmune disease, infection, allergy,exposure to irritants in a variety of tissues, and is thought to play animportant role in the pathogenesis of numerous disorders (e.g. migraine,arthritis, rhinitis, gastritis, colitis, cystitis, and sunburn). One wayto reduce neurogenic inflammation is to block excitability innociceptors, thereby preventing the activation of nociceptor peripheralterminals and the release of pro-inflammatory chemicals.

Despite the development of a variety of therapies for pain, itch, andneurogenic inflammation, there is a need for additional agents.

SUMMARY OF THE INVENTION

The present invention provides compounds represented by Formula (I) thatcan be used to treat or prevent pain, itch, and neurogenic inflammation:

wherein

Y⁻ is a pharmaceutically acceptable anion;

R^(F) and R^(G) together with N⁺ form an optionally substitutedheteroaryl ring having one or more heteroatoms or an optionallysubstituted bicyclic heteroaryl ring having one or more heteroatoms;

R^(A), R^(B), and R^(C) are each independently selected from H, D,halogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, nitrile,OR^(I), NR^(J)R^(K), NR^(L)C(O)R^(M), S(O)R^(N), SO₂R^(O),SO₂R^(O)R^(P), SO₂NR^(Q)R^(R), SO₃R^(S), CO₂R^(T), C(O)R^(U), andC(O)NR^(V)R^(W); or R^(B) and vicinal R^(C) together with the carbonatoms to which they are attached form a substituted or unsubstituted3-7-membered cycloalkyl (a C₃-C₇ cycloalkyl) or a substituted orunsubstituted aryl (for example, a phenyl);

each of R^(I), R^(J), R^(K), R^(L), R^(M), R^(N), R^(O), R^(P), R^(O),R^(R), R^(S), R^(T), R^(U), R^(V), and R^(W) is independently selectedfrom H, D, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, andsubstituted or unsubstituted heteroalkyl;

X¹ is selected from —CR^(X)R^(Y)—, —NR^(Z)C(O)—, —OC(O)—, —SC(O)—,—NR^(Z)S(O)—, —S(O)NR^(Z)—, —NR^(X)C(O)NR^(Y)—, —C(O)NR^(1A)—, —C(O)O—,—C(O)—, —S(O)—, —S(O)₂—, and —(O)CS—; X¹ can also be—NR^(Z)C(O)CR^(X)R^(Y)—,

each of R^(X), R^(Y), R^(Z), and R^(1A) is independently selected fromH, D, substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, and substituted orunsubstituted heteroalkyl; and

each of R^(D) and R^(E) is independently selected from H, D, substitutedor unsubstituted alkyl, substituted or unsubstituted alkenyl,substituted or unsubstituted alkynyl, substituted or unsubstitutedheteroalkyl, optionally substituted with halogen, cyclic alkyl, aryl, orheteroaryl, and cycloalkyl; or R^(D) and R^(E) together with the carbonto which they are attached to form a substituted or unsubstituted3-7-membered cycloalkyl (a C₃-C₇ cycloalkyl) or a substituted orunsubstituted heterocyclic or heteroalkyl ring; or R^(D) and R^(Z)together with the carbon and the —N—C(O)— to which they are attachedform an optionally substituted 5-8-membered lactam.

In a preferred embodiment, X¹ is —NHC(O)— or —C(O)NH—. In additionalpreferred embodiments, X¹ is —NHC(O)—.

In some embodiments, each of R^(A) and R^(B) is independently selectedfrom H, D, halogen, substituted or unsubstituted C₁₋₄ (C₁-C₄) alkyl, andNR^(J)R^(K); and each of R^(J) and R^(K) is independently selected fromH and substituted or unsubstituted C₁₋₄ alkyl; and/or wherein R^(C) isnot H, such as halogen, C₁₋₄ alkyl, and NR^(J)R^(K).

In a preferred embodiment, each of R^(A) and R^(B) is —CH₃.

In certain other embodiments, R^(D) is C₁₋₄ alkyl optionally substitutedwith a substituent selected from the group consisting of halogen, oxygen(oxo), C₃₋₈ (C₃-C₈) cycloalkyl, aryl, and heteroaryl, and/or R^(E) is Hor C₁₋₄ alkyl optionally substituted with a substituent selected fromthe group consisting of halogen, oxygen, C₃₋₈ cycloalkyl, aryl, andheteroaryl.

In preferred embodiments, each of R^(D) and R^(E) is independentlyselected from —H, —CH₃, —CH₂CH₃, and —(CH₂)₂CH₃. In a more preferredembodiment, R^(E) is hydrogen and R^(D) is —H, —CH₃, —CH₂CH₃, or—(CH₂)₂CH₃.

In certain preferred embodiments, R^(D) and R^(E) are both hydrogen. Inyet additional preferred embodiments, R^(D) is hydrogen and R^(E) is analkyl, for example, a C₁-C₆ alkyl or a C₁-C₄ alkyl including, but notlimited to, methyl, ethyl, propyl and butyl. In certain additionalpreferred embodiments, R^(D) and R^(E) are taken together with thecarbon to which they are attached to form a C₃-C₆ cycloalkyl including,but not limited to, cyclopropyl or cyclobutyl.

In some embodiments Y⁻ includes, but is not limited to, a halide ion, asubstituted or unsubstituted alkyl sulfonate, a substituted orunsubstituted aryl sulfonate, an aliphatic carboxylate, a substitutedaliphatic carboxylate, an aryl carboxylate, a substituted arylcarboxylate, a heterocyclyl carboxylate or a substituted heterocyclylcarboxylate.

In additional aspects, Y⁻ is a halide ion. In one embodiment, Y⁻ is thehalide ion selected from bromide, chloride, and iodide.

Each embodiment stated herein can be taken in combination with one, anyor all other embodiments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds represented by Formula (I) asdescribed above, or pharmaceutically acceptable salts, stereoisomers,solvates, hydrates or combinations thereof. The invention also providescompositions comprising compounds having Formula (I) or apharmaceutically acceptable salts thereof, for example, a compositioncomprising an effective amount of a compound of Formula (I) orpharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable excipient. The compositions of the invention may furthercomprise compounds of the invention and a biologically active agent. Thecompositions described herein can be formulated for oral, intravenous,intramuscular, rectal, cutaneous, subcutaneous, topical, transdermal,sublingual, nasal, inhalation, vaginal, intrathecal, epidural, or ocularadministration.

The invention further provides methods for treating pain, cough, itch,or a neurogenic inflammatory disorder in a patient, includingadministering to the patient a composition comprising a compound havingFormula (I), wherein the compound inhibits one or more voltage-gated ionchannels present in nociceptors and/or cough receptors and/orpruriceptors when exposed or applied to the internal face of thechannels but does not substantially inhibit the channels when applied tothe external face of the channels, and wherein the compound is capableof entering nociceptors, cough receptors or pruriceptors through a largepore channel when the channel is activated and inhibiting one or morevoltage-gated ion channels present in the nociceptors, cough receptorsor pruriceptors.

In certain embodiments, the large pore channel is a transient receptorpotential ion channel (TRP channel). In other embodiments, the TRPchannel is activated by an exogenous or endogenous agonist. In yet otherembodiments, the large pore channel is TRPA1, TRPV1-4, TRPM8, ASIC orP2X. In particular embodiments, the compound is capable of enteringnociceptors, cough receptors or pruriceptors through the TRPA1, TRPV1-4,TRPM8, ASIC or P2X receptor/channel when the receptor/channel isactivated. In yet another embodiment, the compound inhibitsvoltage-gated sodium channels. In yet another embodiment, the type ofpain treated by the methods, compositions, and kits of the invention isselected from the group consisting of neuropathic pain, inflammatorypain, nociceptive pain, pain due to infections, and procedural pain, orwherein the neurogenic inflammatory disorder is selected from the groupconsisting of allergic inflammation, asthma, chronic cough,conjunctivitis, rhinitis, psoriasis, inflammatory bowel disease,interstitial cystitis, and atopic dermatitis.

We have identified compounds having Formula (I):

that are capable of passing through open large pore channels that areexpressed on nociceptors and/or cough receptors and/or pruriceptors butnot on motor neurons. Because the ion channel blocking compounds of thepresent invention are positively charged, they are notmembrane-permeable and thus cannot enter cells that do not express largepore channels. Since large pore channels are often more active in tissueconditions associated with pain (such as inflammation) due to release ofendogenous ligands or activation by thermal stimuli, the ion channelblocker of the invention can be used alone to selectively targetactivated nociceptors in order to effectively treat (e.g., eliminate oralleviate) pain, cough, itch, or neurogenic inflammation. The ionchannel blockers of the invention can also be used in combination withone or more exogenous large pore receptor agonists to selectively targetnociceptors in order to effectively treat (e.g., eliminate or alleviate)pain, cough, itch, or neurogenic inflammation.

Voltage-dependent ion channels in pain-sensing neurons are currently ofgreat interest in developing drugs to treat pain. Blockingvoltage-dependent sodium channels in pain-sensing neurons can block painsignals by interrupting initiation and transmission of the actionpotential. Moreover, blocking voltage-dependent sodium channels innociceptors can reduce or eliminate neurogenic inflammation bypreventing activation of nociceptor peripheral terminals and the releasethereof pro-inflammatory chemicals.

Heretofore, a limitation in treating with molecules that block sodiumchannels or calcium channels is that the vast majority of suchexternally-applied molecules are hydrophobic and can pass throughmembranes. Because of this, they will enter all cells and thus have noselectivity for affecting only nociceptors.

The inhibitors of the present invention are membrane-impermeable and areonly effective when present inside the nociceptor cell, and thus mustpass through the cell membrane via a channel or receptor, such as largepore channels (e.g., TRPAV1-4, TRPA1, TRPM8, ASIC and P2X(2/3)), inorder to produce an effect. Under normal circumstances, most large porechannels in nociceptors are not active but require a noxious thermal,mechanical, or chemical stimulus to activate them. For example, TRPchannels in nociceptors can be activated by an exogenous TRP ligand(i.e. TRP agonist) such as capsaicin, which opens the TRPV1 channel.Thus, one approach to selectively targeting nociceptors is toco-administer the membrane-impermeable ion channel inhibitor with anexogenous TRP ligand that permits passage of the inhibitor through theTRP channel into the cell. In addition to capsaicin, the exogenous TRPligand can also be another capsaicinoid, mustard oil, or lidocaine. Inanother example, TRP channels may be active in response to exogenousirritant activators such as inhaled acrolein from smoke or chemicalwarfare agents such as tear gas.

Under certain circumstances, large pore channels can be activated in theabsence of exogenous large pore channel agonists/ligands by endogenousinflammatory activators that are generated by tissue damage, infection,autoimmunity, atopy, ischemia, hypoxia, cellular stress, immune cellactivation, immune mediator production, and oxidative stress. Under suchconditions, endogenous molecules (e.g., protons, lipids, and reactiveoxygen species) can activate large pore channels expressed onnociceptors, allowing membrane-impermeable, voltage-gated ion channelblockers to gain access to the inside of the nociceptor through theendogenously-activated large pore channels. Endogenous inflammatoryactivators of large pore channels include, for example, prostaglandins,nitric oxide (NO), peroxide (H₂O₂), cysteine-reactive inflammatorymediators like 4-hydroxynonenal, protons, ATP, endogenous alkenylaldehydes, endocannabinoids, and immune mediators (e.g., interleukin 1(IL-1), nerve growth factor (NGF), and bradykinin, whose receptors arecoupled to large pore channels).

Definitions

As used herein, the words “a” and “an” are meant to include one or moreunless otherwise specified.

By “biologically active” is meant that a molecule, including biologicalmolecules, such as nucleic acids, peptides, polypeptides, and proteins,exerts a biological, physical or chemical effect or activity on aprotein, enzyme, receptor, ligand, antigen, itself or other molecule.For example, a “biologically active” molecule may possess, e.g.,enzymatic activity, protein binding activity, or pharmacologicalactivities.

Biologically active agents that can be used in the methods and kitsdescribed herein include, without limitation, TRPA1 receptor agonists,TRPV1-4 receptor agonists, ASIC agonists, TRPM8 agonists, P2X receptoragonists, NSAIDs, glucocorticoids, narcotics, anti-proliferative andimmune modulatory agents, an antibody or antibody fragment, anantibiotic, a polynucleotide, a polypeptide, a protein, an anti-canceragent, a growth factor, and a vaccine.

By “inflammation” is meant any types of inflammation, such those causedby the immune system (immune-mediated inflammation) and by the nervoussystem (neurogenic inflammation), and any symptom of inflammation,including redness, heat, swelling, pain, and/or loss of function.

By “neurogenic inflammation” is meant any type of inflammation mediatedor contributed to by neurons (e.g. nociceptors) or any other componentof the central or peripheral nervous system.

The term “pain” is used herein in the broadest sense and refers to alltypes of pain, including acute and chronic pain, such as nociceptivepain, e.g., somatic pain and visceral pain; inflammatory pain,dysfunctional pain, idiopathic pain, neuropathic pain, e.g., centrallygenerated pain and peripherally generated pain, migraine, and cancerpain.

The term “nociceptive pain” is used to include all pain caused bynoxious stimuli that threaten to or actually injure body tissues,including, without limitation, by a cut, bruise, bone fracture, crushinjury, burn, and the like. Pain receptors for tissue injury(nociceptors) are located mostly in the skin, musculoskeletal system, orinternal organs.

The term “somatic pain” is used to refer to pain arising from bone,joint, muscle, skin, or connective tissue. This type of pain istypically well localized.

The term “visceral pain” is used herein to refer to pain arising fromvisceral organs, such as the respiratory, gastrointestinal tract andpancreas, the urinary tract and reproductive organs. Visceral painincludes pain caused by tumor involvement of the organ capsule. Anothertype of visceral pain, which is typically caused by obstruction ofhollow viscus, is characterized by intermittent cramping and poorlylocalized pain. Visceral pain may be associated with inflammation as incystitis or reflux esophagitis.

The term “inflammatory pain” includes pain associates with activeinflammation that may be caused by trauma, surgery, infection andautoimmune diseases.

The term “neuropathic pain” is used herein to refer to pain originatingfrom abnormal processing of sensory input by the peripheral or centralnervous system consequent on a lesion to these systems.

The term “procedural pain” refers to pain arising from a medical, dentalor surgical procedure wherein the procedure is usually planned orassociated with acute trauma.

The term “itch” is used herein in the broadest sense and refers to alltypes of itching and stinging sensations localized and generalized,acute intermittent and persistent. The itch may be idiopathic, allergic,metabolic, infectious, drug-induced, due to liver, kidney disease, orcancer. “Pruritus” is severe itching.

The term “cough” is used herein to refer to an aberrant cough reflexresulting in chronic non-productive dry cough and which is manifested bya hyper- or allo-tussive state. This cough may be found in a number ofdisease states including asthma, COPD, asthma-COPD overlap syndrome(ACOS), interstitial pulmonary fibrosis (IPF) and lung cancer. Inaddition, inappropriate cough reflexes can be manifested acutely andchronically following viral infection. Furthermore, chronic cough can beidiopathic in nature with unknown etiology.

By “patient” is meant any animal. In one embodiment, the patient is ahuman. Other animals that can be treated using the methods,compositions, and kits of the invention include but are not limited tonon-human primates (e.g., monkeys, gorillas, chimpanzees), domesticatedanimals (e.g., horses, pigs, goats, rabbits, sheep, cattle, llamas), andcompanion animals (e.g., guinea pigs, rats, mice, lizards, snakes, dogs,cats, fish, hamsters, and birds).

Compounds useful in the invention include, but are not limited to, thosedescribed herein in any of their pharmaceutically acceptable forms,including isomers such as diastereomers and enantiomers, salts, esters,amides, thioesters, solvates, and polymorphs thereof, as well as racemicmixtures and pure isomers of the compounds described herein. The term“pharmaceutically acceptable anion” as used herein, refers to theconjugate base of a pharmaceutically acceptable acid. Such acids aredescribed in Stahl, P. H. and Wermuth, C. G. (eds.), Handbook ofPharmaceutical Salts: Properties, Selection and Use, Wiley VCH (2008).Pharmaceutically acceptable acids include, but are not limited to,acetic acid, dichloroacetic acid, adipic acid, alginic acid, L-ascorbicacid, L-aspartic acid, benzenesulfonic acid, 4-acetamidobenzoic acid,benzoic acid, p-bromophenylsulfonic acid, (+)-camphoric acid,(+)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid,carbonic acid, cinnamic acid, cyclamic acid, dodecylsulfuric acid,ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonicacid, sulfuric acid, boric acid, citric acid, formic acid, fumaric acid,galactaric acid, gentisic acid, D-glucoheptonic acid, D-gluconic acid,D-glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid,glycerophosphoric acid, glycolic acid, hippuric acid, hydrochloric acid,hydrobromic acid, hydroiodic acid, isobutyric acid, DL-lactic acid,lactobionic acid, 1 auric acid, maleic acid, (−)-L-malic acid, malonicacid, DL-mandelic acid, methanesulfonic acid, naphthalene-1,5-disulfonicacid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinicacid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid,pamoic acid, phosphoric acid, propionic acid, (−)-L-pyroglutamic acid,salicyclic acid, 4-aminosalicyclic acid, sebacic acid, stearic acid,succinic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonicacid, and undecylenic acid. Pharmaceutically acceptable anions includethe conjugate base of any the acids set forth above.

The term “pharmaceutically acceptable salt” represents those salts whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of humans and lower animals without unduetoxicity, irritation, allergic response and the like, and arecommensurate with a reasonable benefit/risk ratio. The salts can beprepared in situ during the final isolation and purification of thecompounds of the invention, or separately by reacting the free basefunction with a suitable organic acid. Representative acid salts includebut are not limited to acetate, adipate, alginate, ascorbate, aspartate,benzenesulfonate, benzoate, hi sulfate, borate, butyrate, camphorate,camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate,hemi sulfate, heptonate, hexanoate, hydrobromide, hydrochloride,hydroiodide, 2-hydroxy-ethanesulfonate, isethionate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate, mesylate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, pi crate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts,and the like.

In the generic descriptions of compounds of this invention, the numberof atoms of a particular type in a substituent group is generally givenas a range, e.g., an alkyl group containing from 1 to 4 carbon atoms orC₁₋₄ alkyl or C₁-C₄ alkyl. Reference to such a range is intended toinclude specific references to groups having each of the integer numberof atoms within the specified range. For example, an alkyl group from 1to 4 carbon atoms includes each of C₁, C₂, C₃, and C₄ alkyls. Othernumbers of atoms and other types of atoms may be indicated in a similarmanner.

“D” is deuterium.

As used herein, the terms “alkyl” and the prefix “alk-” are inclusive ofboth straight chain and branched chain groups and of cyclic groups,i.e., cycloalkyl. Cyclic groups can be monocyclic or polycyclic andpreferably have from 3 to 6 ring carbon atoms or 3 to 7 carbon atoms,inclusive. Exemplary cyclic groups include cyclopropyl, cyclobutyl,cyclopentyl, and cyclohexyl groups.

By “C₁₋₄ alkyl” or “C₁-C₄ alkyl” is meant a branched or unbranchedhydrocarbon group having from 1 to 4 carbon atoms. Similarly, a “C₁₋₆alkyl” or “C₁-C₆” is a branched or unbranched hydrocarbon group havingfrom 1 to 6 carbon atoms. An alkyl, including, for example, a C₁₋₄ alkylor a C₁₋₆ alkyl group may be substituted or unsubstituted. Exemplarysubstituents include, but are not limited to, alkoxy, aryloxy,sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl,perfluoralkyl, amino, alkylamino, disubstituted amino, quaternary amino,alkylcarboxy, and carboxyl groups. Exemplary substituents also includealkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide (F, Cl, Br orI), hydroxyl, fluoroalkyl, perfluoralkyl, oxo, amino, alkylamino,disubstituted amino, quaternary amino, amido, ester, alkylcarboxy,alkoxycarbonyl, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxyl,alkylcarbonyl, arylcarbonyl, alkylthiocarbonyl, phosphate, phosphonato,phosphinato, acylamino (including alkylcarbonylamino, arylcarbonylamino,carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, aryl, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety. C₁₋₄ alkyls include,without limitation, methyl, ethyl, n-propyl, isopropyl, cyclopropyl,cyclopropylmethyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, andcyclobutyl. C₁₋₆ alkyls include, without limitation, methyl, ethyl,n-propyl, isopropyl, cyclopropyl, cyclopropylmethyl, n-butyl, iso-butyl,sec-butyl, tert-butyl, n-pentyl, n-hexyl, cyclobutyl, cyclopentyl, andcyclohexyl.

An example of a substituted alkyl is a heteroalkyl. By “heteroalkyl” ismeant a branched or unbranched alkyl, cycloalkyl, alkenyl, or alkynylgroup having from 1 to 7 or more carbon atoms in addition to 1, 2, 3 or4 heteroatoms independently selected from the group consisting of N, O,S, and P. By “C₁₋₇ heteroalkyl” is meant a branched or unbranched alkyl,alkenyl, or alkynyl group having from 1 to 7 carbon atoms in addition to1, 2, 3 or 4 heteroatoms independently selected from the groupconsisting of N, O, S, and P. Heteroalkyls can include, withoutlimitation, tertiary amines, secondary amines, ethers, thioethers,amides, thioamides, carbamates, thiocarbamates, hydrazones, imines,phosphodiesters, phosphoramidates, sulfonamides, and disulfides. Aheteroalkyl may optionally include monocyclic, bicyclic, or tricyclicrings, in which each ring desirably has three to six members. Theheteroalkyl group may be substituted or unsubstituted. Exemplarysubstituents include alkyl, alkoxy, aryloxy, sulfhydryl, alkylthio,arylthio, halide (F, Cl, Br or I), hydroxyl, fluoroalkyl, perfluoralkyl,oxo, amino, alkylamino, disubstituted amino, quaternary amino, amido,ester, alkylcarboxy, alkoxycarbonyl, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxyl, alkylcarbonyl, arylcarbonyl,alkylthiocarbonyl, phosphate, phosphonato, phosphinato, acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl, andureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, aryl, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety. Examples of C₁₋₇heteroalkyls include, without limitation, methoxymethyl and ethoxyethyl.

An alkenyl is a branched or unbranched hydrocarbon group containing oneor more double bonds. For example, by “C₂₋₆ alkenyl” or “C₂-C₆ alkenyl”is meant a branched or unbranched hydrocarbon group containing one ormore double bonds and having from 2 to 6 carbon atoms. An alkenyl mayoptionally include monocyclic or polycyclic rings, in which each ringdesirably has from three to six members. The alkenyl group may besubstituted or unsubstituted. Exemplary substituents include thosedescribed above for alkyl, and specifically include alkoxy, aryloxy,sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl,perfluoralkyl, amino, alkylamino, disubstituted amino, quaternary amino,alkylcarboxy, and carboxyl groups. C₂₋₆ alkenyls include, withoutlimitation, vinyl, allyl, 2-cyclopropyl-1-ethenyl, 1-propenyl,1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, and2-methyl-2-propenyl.

An alkynyl is a branched or unbranched hydrocarbon group containing oneor more triple bonds. For example, by “C₂₋₆ alkynyl” or “C₂-C₆ alkynyl”is meant a branched or unbranched hydrocarbon group containing one ormore triple bonds and having from 2 to 6 carbon atoms. An alkynyl mayoptionally include monocyclic, bicyclic, or tricyclic rings, in whicheach ring desirably has five or six members. The alkynyl group may besubstituted or unsubstituted. Exemplary substituents those describedabove for alkyl, and specifically include include alkoxy, aryloxy,sulfhydryl, alkylthio, arylthio, halide, hydroxy, fluoroalkyl,perfluoralkyl, amino, alkylamino, disubstituted amino, quaternary amino,alkylcarboxy, and carboxyl groups. C₂₋₆ alkynyls include, withoutlimitation, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and3-butynyl.

By “heterocyclyl,” “heterocyclic,” or “heterocycloalkyl” is meant astable monocyclic or polycyclic (including a bicyclic or a tricyclic)heterocyclic ring which is saturated, partially unsaturated orunsaturated (including heteroaryl or aromatic), and which consists of 2or more carbon atoms and 1, 2, 3 4 or more heteroatoms independentlyselected from N, O, and S and including any bicyclic or polycyclic groupin which any of the above-defined heterocyclic rings is fused to abenzene ring, heteroaryl, cycloalkyl or heterocycloalkyl. In certainaspects, the heterocyclyl is a 3- to 15-membered ring system, a 3- to12-membered ring system, or a 3- to 9-membered ring system. By “C₂₋₆heterocyclyl” is meant a stable 5- to 7-membered monocyclic or 7- to14-membered bicyclic heterocyclic ring which is saturated, partiallyunsaturated or unsaturated (including heteroaryl or aromatic), and whichconsists of 2 to 6 carbon atoms and 1, 2, 3 or 4 heteroatomsindependently selected from N, O, and S and including any bicyclic groupin which any of the above-defined heterocyclic rings is fused to abenzene ring, heteroaryl, cycloalkyl or heterocycloalkyl. Theheterocyclyl or heteroaryl group may be substituted or unsubstituted.Exemplary substituents include substituted or unsubstituted alkyl, aryl,cycloalkyl, heterocycloalkyl, heteroaryl, alkoxy, aryloxy, sulfhydryl,alkylthio, arylthio, halide, hydroxy, fluoroalkyl, perfluoralkyl, amino,alkylamino, disubstituted amino, quaternary amino, alkylcarboxy, andcarboxyl groups. The nitrogen and sulfur heteroatoms may optionally beoxidized. The heterocyclic ring may be covalently attached via anyheteroatom or carbon atom which results in a stable structure, e.g., animidazolinyl ring may be linked at either of the ring-carbon atompositions or at the nitrogen atom. A nitrogen atom in the heterocyclecan be quaternized. Preferably when the total number of S and O atoms inthe heterocycle exceeds 1, then these heteroatoms are not adjacent toone another. Heterocycles include, without limitation, 1H-indazole,2-pyrrolidonyl, 2H,6H-1,5,2-dithiazinyl, 2H-pyrrolyl, 3H-indolyl,4-piperidonyl, 4aH-carbazole, 4H-quinolizinyl, 6H-1,2,5-thiadiazinyl,acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl,benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazalonyl,carbazolyl, 4aH-carbazolyl, b-carbolinyl, chromanyl, chromenyl,cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl,isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl,1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinylperimidinyl,phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl,phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl,piperidinyl, pteridinyl, piperidonyl, 4-piperidonyl, pteridinyl,purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl,pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl,pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl,quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, carbolinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl,thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl,triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl,1,3,4-triazolyl, xanthenyl, β-lactam, γ-lactam and δ-lactam. Preferred 5to 10 membered heterocycles include, but are not limited to, pyridinyl,pyrimidinyl, triazinyl, furanyl, thienyl, thiazolyl, pyrrolyl,pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, tetrazolyl, benzofuranyl,benzothiofuranyl, indolyl, benzimidazolyl, 1H-indazolyl, oxazolidinyl,isoxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl,benzoxazolinyl, quinolinyl, and isoquinolinyl. Preferred 5 to 6 memberedheterocycles include, without limitation, pyridinyl, quinolinyl,pyrimidinyl, triazinyl, furanyl, thienyl, thiazolyl, pyrrolyl,piperazinyl, piperidinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl,and tetrazolyl. Preferred substituents include phenyl, methyl, ethyl,propyl, butyl, chloro, bromo, fluoro and iodo.

By “aryl” is meant an aromatic group having a ring system comprised ofcarbon atoms with conjugated π electrons (e.g., phenyl). A “C₆-C₁₂ aryl”or “C₆-C₁₀ aryl” is an aryl group that has from 6 to 12 carbon atoms or6 to 10 carbon atoms, respectively. Aryl groups may optionally includemonocyclic, bicyclic, or tricyclic rings, in which each ring desirablyhas five or six members. The aryl group may be substituted orunsubstituted. Exemplary substituents include substituted orunsubstituted alkyl, hydroxy, alkoxy, aryloxy, sulfhydryl, alkylthio,arylthio, halide, fluoroalkyl, carboxyl, alkylcarboxy, amino,alkylamino, monosubstituted amino, disubstituted amino, and quaternaryamino groups. A preferred aryl group is phenyl.

By “aralkyl” is meant a substituted or unsubstituted alkyl that issubstituted by a substituted or unsubstituted aryl (including, forexample, (e.g., benzyl, phenethyl, or 3,4-dichlorophenethyl).

By “heteroaralkyl” is meant a substituted or unsubstituted alkyl that issubstituted by or heteroaryl group.

By “C₇₋₁₄ aralkyl” is meant an alkyl substituted by an aryl group (e.g.,benzyl, phenethyl, or 3,4-dichlorophenethyl) having from 7 to 14 carbonatoms.

By “C₃₋₁₀ heterocycloalkyl” is meant an alkyl substituted heterocyclicgroup having from 3 to 10 carbon atoms in addition to one or moreheteroatoms (e.g., 3-furanylmethyl, 2-furanylmethyl,3-tetrahydrofuranylmethyl, or 2-tetrahydrofuranylmethyl).

By “halide” or “halogen” is meant bromine, chlorine, iodine, orfluorine.

By “fluoroalkyl” is meant an alkyl group that is substituted with afluorine atom.

By “alkylcarboxy” is meant a chemical moiety with the formula —(R)—COOH,wherein R is selected from C₁₋₇ alkyl, C₂₋₇ alkenyl, C₂₋₇ alkynyl, C₂₋₆heterocyclyl, C₆₋₁₂ aryl, C₇₋₁₄ aralkyl, C₃₋₁₀ heterocycloalkyl, or C₁₋₇heteroalkyl.

By “alkoxy” is meant a chemical substituent of the formula —OR, whereinR is a substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, or substituted or unsubstituted alkynyl or R can be selectedfrom C₁₋₇ alkyl, C₂₋₇ alkenyl, C₂₋₇ alkynyl, C₂₋₆ heterocyclyl, C₆₋₁₂aryl, C₇₋₁₄ aralkyl, C₃₋₁₀ heterocycloalkyl, or C₁₋₇ heteroalkyl.

By “aryloxy” is meant a chemical substituent of the formula —OR, whereinR is a C₆₋₁₂ aryl group.

By “alkylthio” is meant a chemical substituent of the formula —SR,wherein R is selected from C₁₋₇ alkyl, C₂₋₇ alkenyl, C₂₋₇ alkynyl, C₂₋₆heterocyclyl, C₆₋₁₂ aryl, C₇₋₁₄ aralkyl, C₃₋₁₀ heterocycloalkyl, or C₁₋₇heteroalkyl.

By “arylthio” is meant a chemical substituent of the formula —SR,wherein R is a C₆₋₁₂ aryl group.

By “charged moiety” is meant a moiety which gains a proton atphysiological pH thereby becoming positively charged (e.g., ammonium,guanidinium, or amidinium) or a moiety that includes a net formalpositive charge without protonation (e.g., quaternary ammonium). Thecharged moiety may be either permanently charged or transiently charged.

By “therapeutically effective amount” or “effective amount” means anamount sufficient to produce a desired result, for example, thereduction or elimination of pain, cough, itch, or neurogenicinflammation in a patient (e.g., a human) suffering from a condition,disease, or illness that is caused wholly or in part by neurogenicinflammation (e.g., asthma, arthritis, colitis, contact dermatitis,diabetes, eczema, cystitis, chronic refractory cough, post-viral cough,gastritis, migraine headache, psoriasis, rhinitis, rosacea, or sunburn).

“Solvates” means solvent addition forms that contain eitherstoichiometric or nonstoichiometric amounts of solvent.

The compounds of the present invention, including salts of thecompounds, can exist in unsolvated forms as well as solvated forms,including hydrated forms and unhydrated forms. In general, the solvatedforms are equivalent to unsolvated forms and are encompassed within thescope of the present invention. Nonlimiting examples of hydrates includemonohydrates, dihydrates, hemihydrates, etc. In certain aspects, thecompound is a hemihydrate. Nonlimiting examples of solvates includeethanol solvates, acetone solvates, etc.

The compounds of the invention may exist in multiple crystalline oramorphous forms. In general, all physical forms are equivalent for usescontemplated by the present invention and are intended to be within thescope of the invention.

Compounds that can be used in the compositions, kits, and methods of theinvention include compounds having Formula (I), or a pharmaceuticallyacceptable salt thereof:

wherein

Y⁻ is a pharmaceutically acceptable anion;

R^(F) and R^(G) together with the N⁺ to which they are attached form anoptionally substituted heteroaryl ring having zero, one, or moreheteroatoms in addition to the N⁺, or an optionally substituted bicyclicheteroaryl ring having zero, one, or more heteroatoms in addition to theN⁺;

R^(A), R^(B), and R^(C) are each independently selected from H, D,halogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, nitrile,OR^(I), NR^(J)R^(K), NR^(L)C(O)R^(M), S(O)R^(N), SO₂R^(O),SO₂R^(O)R^(P), SO₂NR^(Q)R^(R), SO₃R^(S), CO₂R^(T), C(O)R^(U), andC(O)NR^(V)R^(W); or R^(B) and vicinal R^(C) together with the carbonatoms to which they are attached form a substituted or unsubstituted3-7-membered cycloalkyl (a C₃-C₇ cycloalkyl) or a substituted orunsubstituted aryl (for example, a phenyl);

each of R^(I), R^(J), R^(K), R^(L), R^(M), R^(N), R^(O), R^(P), R^(Q),R^(R), R^(S), R^(T), R^(U), R^(V), and R^(W) is independently selectedfrom H, D, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, and substituted or unsubstituted alkynyl;

X¹ is selected from —CR^(X)R^(Y)—, —NR^(Z)C(O)—,—NR^(Z)C(O)CR^(X)R^(Y)—, —OC(O)—, —SC(O)—, —NR^(Z)S(O)—, —S(O)NR^(Z)—,—NR^(X)C(O)NR^(Y)—, —C(O)NR^(1A)—, —C(O)O—, —(O)CS—, —S(O)—, —S(O)₂—,and —C(O)—;

each of R^(X), R^(Y), R^(Z), and R^(1A) is independently selected fromH, D, substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, and substituted or unsubstituted alkynyl; and

each of R^(D) and R^(E) is independently selected from H, D, substitutedor unsubstituted alkyl, substituted or unsubstituted alkenyl,substituted or unsubstituted alkynyl, cycloalkyl (for example, a C₃-C₆or C₃-C₇ cycloalkyl cycloalkyl), aryl, or heteroaryl, or R^(U) and R^(E)together with the carbon to which they are attached to form asubstituted or unsubstituted C₃-C₇ cycloalkyl or a substituted orunsubstituted heterocyclic ring (for example, a 5- to 7-memberedheterocyclic ring), or R^(D) and R^(Z) together with the carbon and the—N—C(O)— to which they are attached form an optionally substituted5-8-membered lactam.

In some embodiments, the heteroaryl ring or bicyclic heteroaryl ringformed by R^(F) and R^(G) together with the N⁺ to which they areattached may be optionally substituted with substituted or unsubstitutedCue alkane, Cue heteroalkane, carbocycle, substituted carbocycle,heterocarbocycle, substituted heterocarbocycle, phenyl, substitutedphenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,carboxamide, hydroxy, ether, amide, ester, sulfonamide, sulfone, amino,amino alkyl, urea, nitrile, or halogen. In certain aspects, theheteroaryl ring or bicyclic heteroaryl ring formed by R^(F) and R^(G)together with the N⁺ may be unsubstituted. In additional aspects, theheteroaryl ring or bicyclic heteroaryl ring formed by R^(F) and R^(G)together with the N⁺ may be substituted with a substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted C₃-C₆cycloalkyl, substituted and unsubstituted 3- to 15-memberedheterocyclyl, alkoxy, or CO₂R^(2A), wherein R^(2A) is selected from H,D, substituted or unsubstituted alkyl for example a substituted orunsubstituted C₁-C₆ alkyl), and substituted or unsubstituted alkenyl(for example, a C₂-C₆ alkenyl). In yet additional preferred aspects, theheteroaryl ring or bicyclic heteroaryl ring formed by R^(F) and R^(G)together with the W may be substituted with a substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted C₃-C₆cycloalkyl, substituted and unsubstituted 3- to 8-membered heterocyclyl,OR^(2B), or CO₂R^(2A), wherein R^(2A) is selected from H or substitutedor unsubstituted C₁-C₆ alkyl and R^(2B) is substituted or unsubstitutedC₁-C₆ alkyl. In further aspects, the heteroaryl ring or bicyclicheteroaryl ring formed by R^(F) and R^(G) together with the N⁺ may besubstituted with a substituted or unsubstituted 3- to 8-memberedheterocyclyl, wherein the 3- to 8-membered heterocyclyl contains atleast one nitrogen ring atom.

In a preferred embodiment, the heteroaryl ring or bicyclic heteroarylring formed by R^(F) and R^(G) together with the N⁺ is substituted witha C₁₋₆ alkane or C₁-C₆ alkyl is selected from methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl,neopentyl, and n-hexyl. In a preferred embodiment, the heteroaryl ringor bicyclic heteroaryl ring formed by R^(F) and R^(G) together with theN⁺ is substituted with a group selected from —O-methyl, —O— ethyl,—O-propyl, —O-isopropyl, —O-butyl, —O-isobutyl, —O-cyclohexyl,—O-cyclopentyl, and -ethyl-O-methyl. In a preferred embodiment, theheteroaryl ring or bicyclic heteroaryl ring formed by R^(F) and R^(G)together with the N⁺ is substituted with a carbocycle or C₃-C₆cycloalkyl selected from cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl. In a preferred embodiment, the heteroaryl ring or bicyclicheteroaryl ring formed by R^(F) and R^(G) together with the N⁺ issubstituted with a heterocarbocycle or heterocyclic selected fromaziridine, azetidine, furan, pyrrolidine, pyran, piperidine, piperazine,azepine, and diazapine. In yet further aspects, the heteroaryl ring orbicyclic heteroaryl ring formed by R^(F) and R^(G) together with the N⁺is substituted with a heterocarbocycle or heterocyclic selected fromthose shown in Tables 2 to 6.

In a preferred embodiment, the heteroaryl ring or bicyclic heteroarylring formed by R^(F) and R^(G) together with N⁺ may be optionallysubstituted with methylphenyl, pyridinyl, methylpyridinyl, —C(O)NH₂,—C(O)N(CH₃)₂, —C(O)OCH₃, —C(O)OCH₂CH₃, —S(O)₂NH₂, —S(O)₂N(CH₃)₂,—S(O)₂CH₃, —NHS(O)₂CH₃, —NHC(O)CH₃, —C(O)N(CH₃)₂, —O(CH₂)₂₀CH₃,fluorine, chlorine, tetrazolyl, methylpyrrolidinyl, methylamino,pyrrolidine, and dimethylamino.

In a preferred embodiment, X¹ is —NHC(O)— or —C(O)NH—. In anotherpreferred embodiment, X¹ is —NHC(O)—.

In some embodiments, each of R^(A) and R^(B) is independently selectedfrom H, D, halogen, substituted or unsubstituted C₁₋₄ alkyl, andNR^(J)R^(K); and each of R^(J) and R^(K) is independently selected fromH and substituted or unsubstituted C₁₋₄ alkyl; and/or wherein R^(C) isnot H, such as halogen, substituted or unsubstituted C₁₋₄ alkyl, andNR^(J)R^(K).

In yet additional preferred aspects, R^(A), R^(B), and R^(C) areindependently selected from H, D, halogen, OR^(I), substituted orunsubstituted C₁-C₄ alkyl, and NR^(J)R^(K); wherein each of R^(I), R^(J)and R^(K) is independently selected from H and substituted orunsubstituted C₁-C₄ alkyl. In a preferred embodiment, each of R^(A) andR^(B) is CH₃, and R^(C) is selected from the group consisting of H, CH₃,halogen, nitrile (cyano), methoxy, and ethoxy. In further preferredembodiments, each of R^(A) and R^(B) is CH₃, and R^(C) is selected fromthe group consisting of H, CH₃, fluoro, chloro, nitrile, methoxy, andethoxy. In additional preferred embodiments, each of R^(A) and R^(B) areCH₃ and R^(C) is hydrogen.

In a preferred embodiment, each of R^(A) and R^(B) is —CH₃.

In certain aspects, or R^(B) and vicinal R^(C) together with the carbonatoms to which they are attached form a substituted or unsubstituted3-7-membered cycloalkyl (a C₃-C₇ cycloalkyl) or a substituted orunsubstituted aryl (for example, a phenyl).

In certain other embodiments, R^(D) is C₁₋₄ alkyl optionally substitutedwith a substituent selected from the group consisting of halogen,oxygen, C₃₋₈ cycloalkyl, aryl, and heteroaryl and/or R^(E) is H or C₁₋₄alkyl optionally substituted with a substituent selected from the groupconsisting of halogen, oxygen, C₃₋₈ cyclic alkyl, aryl, or heteroaryl.

In preferred embodiments, each of R^(D) and R^(E) is independentlyselected from —H, —CH₃, —CH₂CH₃, and —(CH₂)₂CH₃. In a more preferredembodiment, R^(E) is hydrogen and R^(D) is —H, —CH₃, —CH₂CH₃, or—(CH₂)₂CH₃.

In certain preferred embodiments, R^(D) and R^(E) are both hydrogen. Inyet additional preferred embodiments, R^(D) is hydrogen and R^(E) is analkyl, for example, a C₁-C₆ alkyl or a C₁-C₄ alkyl including, but notlimited to, methyl, ethyl, propyl and butyl. In certain additionalpreferred embodiments, R^(D) and R^(E) are taken together with thecarbon to which they are attached to form a C₃-C₆ cycloalkyl including,but not limited to, cyclopropyl or cyclobutyl.

In some embodiments Y⁻ is a halide anion, a carboxylate, or a sulfonate.Y⁻ can, for example, be a halide ion, a substituted or unsubstitutedalkylsulfonate, a substituted or unsubstituted arylsulfonate, asubstituted or unsubstituted alkyl or aliphatic carboxylate, asubstituted or unsubstituted aryl carboxylate, or a substituted orunsubstituted heterocyclyl carboxylate.

In certain embodiments, Y− is selected from the group consisting oftrifluoroacetate, sulfate, phosphate, acetate, fumarate, formate,carbonate, maleate, citrate, pyruvate, succinate, oxalate, a sulfonate,(for example, methanesulfonate, trifluoromethanesulfonate,toluenesulfonate such as p-toluenesulfonate, benzenesulfonate,ethanesulfonate, camphorsulfonate, 2-mesitylenesulfonate, ornaphthalenesulfonate such as 2-naphthalenesulfonate), bisulfate,malonate, xinafoate, ascorbate, oleate, nicotinate, saccharinate,adipate, formate, glycolate, L-lactate, D-lactate, aspartate, malate,L-tartrate, D-tartrate, stearate, 2-furoate, 3-furoate, napadisylate(naphthalene-1,5-disulfonate or naphthalene-1-(sulfonicacid)-5-sulfonate), edisylate (ethane-1,2-disulfonate orethane-1-(sulfonic acid)-2-sulfonate), isethionate(2-hydroxyethylsulfonate), D-mandelate, L-mandelate, propionate,tartarate, phthalate, hydrochlorate, hydrobromate, and nitrate. In oneembodiment, Y− is a halide anion.

In one embodiment Y⁻ is the halide ion selected from bromide, chlorideand iodide.

Each embodiment stated herein can be taken in combination with one, anyor all other embodiments.

In certain preferred aspects, the present invention relates to compoundsof Formula (I), or a pharmaceutically acceptable salt thereof, whereinR^(F) and R^(G) together with N⁺ form an optionally substitutedpyridinium ring. In additional aspects, the pyridinium ring formed byR^(F) and R^(G) together with the N⁺ is unsubstituted. In yet additionalaspects, the pyridinium formed by R^(F) and R^(G) together with the N⁺may be substituted with a substituent selected from the group consistingof substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted C₃-C₆ cycloalkyl, substituted and unsubstituted 3- to15-membered heterocyclyl, alkoxy, and CO₂R^(2A) wherein R^(2A) isselected from H, D, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl. In yet additional preferred aspects, thepyridinium ring formed by R^(F) and R^(G) together with the N⁺ may besubstituted with a substituent selected from the group consisting ofsubstituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstitutedC₃-C₆ cycloalkyl, substituted and unsubstituted 3- to 8-memberedheterocyclyl, OR2^(B), or CO₂R^(2A) wherein R^(2A) is selected from H orsubstituted or unsubstituted C₁-C₆ alkyl and R^(2B) is substituted orunsubstituted C₁-C₆ alkyl. In further aspects, the pyridinium ringformed by R^(F) and R^(G) together with the N⁺ may be substituted with asubstituted or unsubstituted 3- to 8-membered heterocyclyl, wherein the3- to 8-membered heterocyclyl contains a nitrogen ring atom.

In a preferred embodiment, the present invention relates to compounds ofFormula (I), or a pharmaceutically acceptable salt thereof, whereinR^(F) and R^(G) together with the N⁺ form an optionally substitutedheteroaryl ring comprising one or more nitrogens selected from one ofthe following:

wherein each of the above shown monocyclic heteroaryl is optionallysubstituted when possible.

In a preferred embodiment, the present invention relates to compounds ofFormula (I), or a pharmaceutically acceptable salt thereof, whereinR^(F) and R^(G) together with the N⁺ form an optionally substitutedbicyclic heteroaryl ring comprising one or more nitrogens, such as:

In yet further preferred aspects, the invention is directed to compoundsof Formula (I), or a pharmaceutically acceptable salt thereof, whereinR^(F) and R^(G) together with the N⁺ form a heteroaryl ring selectedfrom those shown in Tables 1 to 6 below.

In additional embodiments, the invention encompasses a compound ofFormula (II), or a pharmaceutically acceptable salt thereof:

wherein Y⁻ is as defined for Formula (I);

one of R¹ and R² is hydrogen, and the other of R¹ and R² is selectedfrom the group consisting of methyl, ethyl, unsubstituted phenyl, andC(O)OR³; and

R³ is selected from the group consisting of hydrogen, methyl, and ethyl.

In certain aspect, the compound has the Formula (II), wherein Y⁻ is ahalide ion, a sulfonate (including, for example, a substituted orunsubstituted alkylsulfonate and a substituted or unsubstitutedarylsulfonate), carboxylates (including, for example, a substituted orunsubstituted alkyl or aliphatic carboxylate, a substituted orunsubstituted aryl carboxylate, or a substituted or unsubstitutedheterocyclyl carboxylate). In yet further aspect, the compound has theFormula (II), wherein Y⁻ is selected from the group consisting oftrifluoroacetate, sulfate, phosphate, acetate, fumarate, formate,carbonate, maleate, citrate, pyruvate, succinate, oxalate, a sulfonate,(for example, methanesulfonate, trifluoromethanesulfonate,toluenesulfonate such as p-toluenesulfonate, benzenesulfonate,ethanesulfonate, camphorsulfonate, 2-mesitylenesulfonate, ornaphthalenesulfonate such as 2-naphthalenesulfonate), bisulfate,malonate, xinafoate, ascorbate, oleate, nicotinate, saccharinate,adipate, formate, glycolate, L-lactate, D-lactate, aspartate, malate,L-tartrate, D-tartrate, stearate, 2-furoate, 3-furoate, napadisylate(naphthalene-1,5-disulfonate or naphthalene-1-(sulfonicacid)-5-sulfonate), edisylate (ethane-1,2-disulfonate orethane-1-(sulfonic acid)-2-sulfonate), isethionate(2-hydroxyethylsulfonate), D-mandelate, L-mandelate, propionate,tartarate, phthalate, hydrochlorate, hydrobromate, and nitrate. In oneembodiment, Y⁻ is halide anion.

In additional aspects, the compound has the Formula (II), wherein Y⁻ isa halide ion. In one embodiment, Y⁻ is the halide ion selected frombromide, chloride, and iodide.

In certain embodiments, the compound has the Formula (II), wherein R² ishydrogen and R¹ is selected from the group consisting of methyl, ethyl,unsubstituted phenyl, and C(O)OR³, wherein R³ is selected from hydrogen,methyl, and ethyl. In preferred aspects, R¹ is unsubstituted phenyl andR² is hydrogen. In yet additional preferred aspects, R¹ is methyl and R²is hydrogen. In further preferred aspects, R¹ is ethyl and R² ishydrogen. In further preferred aspects, R¹ is C(O)OR³, wherein R³ ismethyl or ethyl, and R² is hydrogen. In additional preferred aspects, R¹is C(O)OR³, wherein R³ is ethyl, and R² is hydrogen.

In further embodiments, the compound has the Formula (II), wherein R¹ ishydrogen and R² is selected from the group consisting of methyl, ethyl,unsubstituted phenyl, and C(O)OR³, wherein R³ is hydrogen, methyl, orethyl. In preferred aspects, R¹ is hydrogen and R² is unsubstitutedphenyl. In yet additional preferred aspects, R¹ is hydrogen and R² ismethyl. In further preferred aspects, R¹ is hydrogen and R² is ethyl. Infurther preferred aspects, R¹ is hydrogen and R² is C(O)OR³, wherein R³is methyl or ethyl. In additional preferred aspects, R¹ is hydrogen andR² is C(O)OR³, wherein R³ is ethyl.

In yet additional embodiments, the invention encompasses a compound ofFormula (III), or a pharmaceutically acceptable salt thereof:

wherein Y⁻ is as defined for Formula (I);

R¹ is selected from the group consisting of methyl, ethyl, propyl(including n-propyl and isopropyl), butyl (including n-butyl, iso-butyl,tert-butyl, and sec-butyl), cyclohexyl, phenyl, and CH₂C(O)NHR₃;

R² is hydrogen or methyl; and

R³ is hydrogen, methyl, or ethyl.

In certain aspects, the compound has the Formula (III), wherein Y⁻ is ahalide ion, a sulfonate (including, for example, a substituted orunsubstituted alkylsulfonate and a substituted or unsubstitutedarylsulfonate), carboxylates (including, for example, a substituted orunsubstituted alkyl or aliphatic carboxylate, a substituted orunsubstituted aryl carboxylate, or a substituted or unsubstitutedheterocyclyl carboxylate). In yet a further aspect, the compound has theFormula (III), wherein Y− is selected from the group consisting oftrifluoroacetate, sulfate, phosphate, acetate, fumarate, formate,carbonate, maleate, citrate, pyruvate, succinate, oxalate, a sulfonate,(for example, methanesulfonate, trifluoromethanesulfonate,toluenesulfonate such as p-toluenesulfonate, benzenesulfonate,ethanesulfonate, camphorsulfonate, 2-mesitylenesulfonate, ornaphthalenesulfonate such as 2-naphthalenesulfonate), bisulfate,malonate, xinafoate, ascorbate, oleate, nicotinate, saccharinate,adipate, formate, glycolate, L-lactate, D-lactate, aspartate, malate,L-tartrate, D-tartrate, stearate, 2-furoate, 3-furoate, napadisylate(naphthalene-1,5-disulfonate or naphthalene-1-(sulfonicacid)-5-sulfonate), edisylate (ethane-1,2-disulfonate orethane-1-(sulfonic acid)-2-sulfonate), isethionate(2-hydroxyethylsulfonate), D-mandelate, L-mandelate, propionate,tartarate, phthalate, hydrochlorate, hydrobromate, and nitrate. In oneembodiment, Y⁻ is halide anion.

In additional aspects, the compound has the Formula (III), wherein Y⁻ isa halide ion. In one embodiment, Y⁻ is the halide ion selected frombromide, chloride, and iodide.

In further aspects, the compound has the Formula (III), wherein R¹ ismethyl and R₂ is hydrogen. In yet additional aspects, the compound hasthe Formula (III), wherein both R₁ and R₂ are methyl.

In yet additional embodiments, the compound has the Formula (III),wherein R₁ is ethyl and R₂ is hydrogen. In other aspects, the compoundhas the Formula (III), wherein R₁ is ethyl and R₂ is methyl.

In further embodiments, the compound has the Formula (III), wherein R₁is n-propyl or iso-propyl, and R₂ is hydrogen. In yet additionalembodiments, the compound has the Formula (III), wherein R₁ is n-propylor iso-propyl, and R₂ is methyl.

In further aspects, the compound has the Formula (III), wherein R₁ isn-butyl, iso-butyl, tert-butyl, or sec-butyl, and R₂ is hydrogen. Infurther aspects, compound has the Formula (III), wherein R₁ is n-butyl,iso-butyl, tert-butyl, or sec-butyl, and R₂ is methyl. In yet additionalaspects, the compound has the Formula (III), wherein R₁ is iso-butyl andR₂ is hydrogen. In further embodiments, the compound has the Formula(III), wherein R₁ is iso-butyl and R₂ is methyl.

In additional embodiments, the compound has the Formula (III), whereinR₁ is cyclohexyl and R₂ is hydrogen. In yet additional aspect, thecompound has the Formula (III), wherein R₁ is cyclohexyl and R₂ ismethyl.

In other embodiments, the compound has the Formula (III), wherein R₁ isphenyl and R₂ is hydrogen. In yet additional aspects, the compound hasthe Formula (III), wherein R₁ is phenyl and R₂ is methyl.

In other embodiments, the compound has the Formula (III), wherein R₁ isCH₂C(O)NHR₃ and R₂ is hydrogen. In additional aspects, the compound hasthe Formula (III), wherein R₁ is CH₂C(O)NHR₃ and R₂ is methyl. Infurther aspects, R₁ is CH₂C(O)NHR₃, R₂ is hydrogen, and R₃ is methyl orethyl. In yet further aspects, R₁ is CH₂C(O)NHR₃, R₂ is methyl, and R₃is methyl or ethyl. In certain additional aspects, R₁ is CH₂C(O)NHR₃, R₂is hydrogen, and R₃ is ethyl. In further aspects, R₁ is CH₂C(O)NHR₃, R₂is methyl, and R₃ is ethyl.

In certain aspects, the compound is selected from Table A below, or apharmaceutically acceptable salt thereof, wherein Y− is apharmaceutically acceptable anion:

TABLE A Compound Chemical Structure Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

Compound 7

Compound 8

Compound 9

Compound 10

Compound 11

Compound 12

Compound 13

Compound 14

Compound 15

Compound 16

Compound 17

Compound 18

Compound 19

Compound 20

Compound 21

Compound 22

Compound 23

Compound 24

Compound 25

Compound 26

Compound 27

Compound 28

Compound 29

Compound 30

In additional preferred aspects, the compound is selected from Table Bbelow, or a pharmaceutically acceptable salt thereof:

TABLE B Compound Y⁻ Chemical Structure Compound 1A Br⁻

Compound 1B Cl⁻

Compound 2A Br⁻

Compound 2B Cl⁻

Compound 3A Br⁻

Compound 3B Cl⁻

Compound 4A Br⁻

Compound 4B Cl⁻

Compound 5A Br⁻

Compound 5B Cl⁻

Compound 6A Br⁻

Compound 6B Cl⁻

Compound 7A Br⁻

Compound 7B Cl⁻

Compound 8A Br⁻

Compound 8B Cl⁻

Compound 9A Br⁻

Compound 9B Cl⁻

Compound 10A Br⁻

Compound 10B Cl⁻

Compound 11A Br⁻

Compound 11B Cl⁻

Compound 12A Br−

Compound 12B Cl⁻

Compound 13A Br⁻

Compound 13B Cl⁻

Compound 14A Br−

Compound 14B Cl⁻

Compound 15A Br−

Compound 15B Cl⁻

Compound 16A Br−

Compound 16B Cl⁻

Compound 17A Br−

Compound 17B Cl⁻

Compound 18A Br−

Compound 18B Cl⁻

Compound 19A Br−

Compound 19B Cl⁻

Compound 20A Br−

Compound 20B Cl⁻

Compound 21A Br−

Compound 21B Cl⁻

Compound 22A Br−

Compound 22B Cl⁻

Compound 23A Br−

Compound 23B Cl⁻

Compound 24A Br−

Compound 24B Cl⁻

Compound 25A Br−

Compound 25B Cl⁻

Compound 26A Br−

Compound 26B Cl⁻

Compound 27A Br−

Compound 27B Cl⁻

Compound 28A Br−

Compound 28B Cl⁻

Compound 29A

Compound 30A Br−

Compound 30B Cl⁻

In certain aspects, the compound is selected from Table C below, or apharmaceutically acceptable salt thereof, wherein Y− is apharmaceutically acceptable anion:

TABLE C Com- pound Structure 31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

In additional preferred aspects, the compound is selected from Table Dbelow, or a pharmaceutically acceptable salt thereof:

TABLE D Compound Y^(—) Structure  31A Cl^(—)

 32A Cl^(—)

 33A Br^(—)

 34A Br^(—)

 35A Br^(—)

 36A Br^(—)

 37A Br^(—)

 38A Br^(—)

 39A Cl^(—)

 40A Cl^(—)

 41A Cl^(—)

 42A Cl^(—)

 43A Cl^(—)

 44A Br^(—)

 45A Br^(—)

 46A Br^(—)

 47A Br^(—)

 48A Br^(—)

 49A Br^(—)

 50A Br^(—)

 51A Br^(—)

 52A Br^(—)

 53A Br^(—)

 54A Br^(—)

 55A Br^(—)

 56A Br^(—)

 57A Br^(—)

 58A Br^(—)

 59A Br^(—)

 60A Br^(—)

 61A Br^(—)

 62A Br^(—)

 63A Br^(—)

 64A Br^(—)

 65A Br^(—)

 66A Br^(—)

 67A Br^(—)

 68A Br^(—)

 69A TFA^(—)

 70A Br^(—)

 71A Br^(—)

 72A Br^(—)

 73A Br^(—)

 74A Br^(—)

 75A Br^(—)

 76A TFA^(—)

 77A Br^(—)

 78A Br^(—)

 79A Br^(—)

 80A TFA^(—)

 81A Br^(—)

 82A Cl^(—)

 83A Br^(—)

 84A Cl^(—)

 85A Br^(—)

 86A Br^(—)

 87A Cl^(—)

 88A Br^(—)

 89A Br^(—)

 90A Br^(—)

 91A Br^(—)

 92A Br^(—)

 93A Br^(—)

 94A Br^(—)

 95A HCOO^(—)

 96A HCOO^(—)

 97A TFA^(—)

 98A Br^(—)

 99A TFA^(—)

100A Br^(—)

101A Br^(—)

102A Br^(—)

103A TFA^(—)

104A HCOO^(—)

105A Br^(—)

In additional preferred aspects, the compound is selected from Table Ebelow, or a pharmaceutically acceptable salt thereof:

TABLE E 106A

107A

108A

109A

110A

111A

112A

113A

114A

115A

116A

117A

118A

119A

120A

121A

122A

123A

124A

125A

126A

127A

128A

129A

Each preferred embodiment described herein can be taken in combinationwith one, any or all other preferred embodiments, as though presentedherein in every permutation.

Compositions of the invention can comprise racemic mixtures, pureenantiomers, or an excess of one enantiomer over the other. For example,the alpha atom of Compound 3 is chiral. Thus, the compound can have thestereochemistry depicted in structures (3a) and (3b):

A composition of the invention can comprise a racemic mixture ofstructure 3a and structure 3b, a pure enantiomer of either structure 3aor structure 3b, or an excess of one enantiomer over the other. Forexample, the composition can comprise the compound in an enantiomericexcess of at least 5, 10, 20, 30, 40, 50, 60, 70, 80 or 90%. In oneembodiment, the enantiomeric excess is at least 95%.

The compounds of the invention include all enantiomers which may bedefined, in terms of absolute stereochemistry, as (R)- or (S)-, as wellas their racemic and optically pure forms, and is not limited to thosedescribed herein in any of their pharmaceutically acceptable forms,including enantiomers, salts, solvates, polymorphs, solvatomorphs,hydrates, anhydrous and other crystalline forms and combinationsthereof. Likewise, all tautomeric forms are intended to be included.

Preferably, a pharmaceutical composition comprises a compound of theinvention as an R enantiomer in substantially pure form; or, apharmaceutical composition comprises a compound of the invention as an Senantiomer in substantially pure form; or, a pharmaceutical compositioncomprises a compound of the invention as enantiomeric mixtures whichcontain an excess of the R enantiomer or an excess of the S enantiomer.It is particularly preferred that the pharmaceutical compositioncontains a compound of the invention which is a substantially pureoptical isomer. For the avoidance of doubt, a compound of the inventioncan, if desired, be used in the form of solvates.

Synthesis

Compounds having Formula (I) can be prepared using methods analogous tothe following general synthetic schemes:

and,

For example,

Compounds of Formula (II) can be prepared using methods analogous tothose described in the Examples and the following general syntheticscheme:

Compounds having Formula (III) can be prepared using methods analogousto that described in the Examples and the following synthetic scheme.

Representative heterocycles that can be coupled with a halide ion suchas the bromide or chloride include, but are not limited to,

Additional Biologically Active Agents and Exogenous Large Pore ChannelAgonists

As described above, the compound or composition of the invention can beadministered with a biologically active agent. For example, one or moreadditional biologically active agents, including those typically used totreat neurogenic inflammation, may be used in combination with acompound or composition of the invention described herein. Thebiologically active agents include, but are not limited to, TRP1Areceptor agonists, TRPV1-4 receptor agonists, TRPM8 agonists, ASICagonists, P2X receptor agonists, acetaminophen, NSAIDs, glucocorticoids,narcotics, tricyclic antidepressants, amine transporter inhibitors,anticonvulsants, anti-proliferative and immune modulatory agents, anantibody or antibody fragment, an antibiotic, a polynucleotide, apolypeptide, a protein, an anti-cancer agent, a growth factor, and avaccine.

TRPV1 agonists that can be employed in the methods, kits andcompositions of the invention include, but are not limited to, any thatactivates TRPV1 receptors on nociceptors and allows for entry of atleast one inhibitor of voltage-gated ion channels (for example, acompound of the invention). A suitable TRPV1 agonist is capsaicin oranother capsaicinoids, which are members of the vanilloid family ofmolecules. Naturally occurring capsaicinoids are capsaicin itself,dihydrocapsaicin, nordihydrocapsaicin, homodihydrocapsaicin,homocapsaicin, and nonivamide. Other suitable capsaicinoids andcapsaicinoid analogs and derivatives for use in the compositions andmethods of the present invention include naturally occurring andsynthetic capsaicin derivatives and analogs including, e.g., vanilloids(e.g., N-vanillyl-alkanedienamides, N-vanillyl-alkanedienyls, andN-vanillyl-cis-monounsaturated alkenamides), capsiate, dihydrocapsiate,nordihydrocapsiate and other capsinoids, capsiconiate,dihydrocapsiconiate and other coniferyl esters, capsiconinoid,resiniferatoxin, tinyatoxin, civamide, N-phenylmethylalkenamidecapsaicin derivatives, olvanil,N-[(4-(2-aminoethoxy)-3-methoxyphenyl)methyl]-9Z-octa-decanamide,N-oleyl-homovanillamide, triprenyl phenols (e.g., scutigeral),gingerols, piperines, shogaols, guaiacol, eugenol, zingerone, nuvanil,NE-19550, NE-21610, and NE-28345. Additional capsaicinoids, theirstructures, and methods of their manufacture are described in U.S. Pat.Nos. 7,446,226 and 7,429,673, which are hereby incorporated byreference.

Additional suitable TRPV1 agonists include but are not limited toeugenol, arvanil (N-arachidonoylvanillamine), anandamide,2-aminoethoxydiphenyl borate (2APB), AM404, resiniferatoxin, phorbol12-phenylacetate 13-acetate 20-homovanillate (PPAHV), olvanil (NE19550), OLDA (N-oleoyldopamine), N-arachidonyldopamine (NADA),6′-iodoresiniferatoxin (6′-IRTX), C18 N-acylethanolamines, lipoxygenasederivatives such as 12-hydroperoxyeicosatetraenoic acid, inhibitorcysteine knot (ICK) peptides (vanillotoxins), piperine, MSK195(N-[2-(3,4-dimethylbenzyl)-3-(pivaloyloxy)propyl]-2-[4-(2-aminoethoxy)-3-methoxyphenyl]acetamide),JYL79(N-[2-(3,4-dimethylbenzyl)-3-(pivaloyloxy)propyl]-N′-(4-hydroxy-3-methoxybenzyl)thiourea),hydroxy-alpha-sanshool, 2-aminoethoxydiphenyl borate, 10-shogaol,oleylgingerol, oleylshogaol, and SU200(N-(4-tert-butylbenzyl)-N′-(4-hydroxy-3-methoxybenzyl)thiourea). Stillother TRPV1 agonists include amylocaine, articaine, benzocaine,bupivacaine, carbocaine, carticaine, chloroprocaine, cyclomethycaine,dibucaine (cinchocaine), dimethocaine (larocaine), etidocaine,hexylcaine, levobupivacaine, lidocaine, mepivacaine, meprylcaine(oracaine), metabutoxycaine, piperocaine, prilocaine, procaine(novacaine), proparacaine, propoxycaine, risocaine, ropivacaine,tetracaine (amethocaine), and trimecaine.

Suitable TRPV2-4 agonists include, but are not limited to, are 2-APB,cannabinol, diphenylboronic anhydride, insulin-like growth factor 1,lysophosphatidylcholine, lysophosphatidylinositol, probenecid,Δ9-tetrahydrocannabinol, vanillin, eugenol, cinnamaldehyde, camphor,carvacrol, thymol, citral, farnesyl diphosphate, tetrahydrocannabivarin,incensole acetate, diphenylboronic anhydride, 6-tert-butyl-m-cresol,dihydrocarveocarveol, borneol, (−)-menthol, GSK1016790A, 4α-PDH,5,6-epoxyeicosatrienoic acid, 4α-PDD, bisandrographolide, citric acid,phorbol 12-myristate 13-acetate and RN1747.

Suitable TRPM8 agonists include, but are not limited to, are menthol,icilin, eucalyptus, linalool, geraniol, hydroxy-citronellal, WS-3,WS-23, Frescolat MGA, Frescolat ML, PMD 38, CPS125, Coolact P, M8-Ag,AITC, cryosim-3 and Cooling Agent 10.

Suitable ASIC agonists include, but are not limited to,chlorophenylguanidine hydrochloride, GMQ hydrochloride,tetrahydropapaveroline (THP), reticulin, polyamine agmatine,lysophosphatidylcholine, arachidonic acid and neuropeptide SF.

Other biologically active agents which can be employed in the methods,compositions, and kits of the invention include any that activates TRP1Areceptors on nociceptors or pruriceptors and allows for entry of atleast one inhibitor of voltage-gated ion channels. Suitable TRP1Aagonists include but are not limited to cinnamaldehyde,allyl-isothiocynanate (mustard oil), diallyl disulfide, icilin, cinnamonoil, wintergreen oil, clove oil, acrolein, hydroxy-alpha-sanshool,2-aminoethoxydiphenyl borate, 4-hydroxynonenal, methylp-hydroxybenzoate, and 3′-carbamoylbiphenyl-3-yl cyclohexylcarbamate(URB597).

P2X agonists that can be employed in the methods, compositions, and kitsof the invention include any that activates P2X receptors on nociceptorsor pruriceptors and allows for entry of at least one inhibitor ofvoltage-gated ion channels. Suitable P2X agonists include but are notlimited to ATP, α,β-methylene ATP, 2-methylthio-ATP, 2′ and3′-O-(4-benzoylbenzoyl)-ATP, and ATP5′-O-(3-thiotriphosphate).

Other biologically active agents that can be used in combination withthe compounds of the invention include NSAIDs, glucocorticoids,narcotics, tricyclic antidepressants, amine transporter inhibitors,anticonvulsants, anti-proliferative and immune modulatory agents, anantibody or antibody fragment, an antibiotic, a polynucleotide, apolypeptide, a protein, an anti-cancer agent, a growth factor, and avaccine.

Non-steroidal anti-inflammatory drugs (NSAIDs) that can be administeredto a patient (e.g., a human) suffering from neurogenic inflammation incombination with a composition of the invention include, but are notlimited to, acetylsalicylic acid, amoxiprin, benorylate, benorilate,choline magnesium salicylate, diflunisal, ethenzamide, faislamine,methyl salicylate, magnesium salicylate, salicyl salicylate,salicylamide, diclofenac, aceclofenac, acemethacin, alclofenac,bromfenac, etodolac, indometacin, nabumetone, oxametacin, proglumetacin,sulindac, tolmetin, ibuprofen, alminoprofen, benoxaprofen, carprofen,dexibuprofen, dexketoprofen, fenbufen, fenoprofen, flunoxaprofen,flurbiprofen, ibuproxam, indoprofen, ketoprofen, ketorolac, loxoprofen,naproxen, oxaprozin, pirprofen, suprofen, tiaprofenic acid, mefenamicacid, flufenamic acid, meclofenamic acid, tolfenamic acid,phenylbutazone, ampyrone, azapropazone, clofezone, kebuzone, metamizole,mofebutazone, oxyphenbutazone, phenazone, sulfinpyrazone, piroxicam,droxicam, lornoxicam, meloxicam, tenoxicam, and the COX-2 inhibitorscelecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, valdecoxib,and pharmaceutically acceptable salts thereof.

Glucocorticoids that can be administered to a patient (e.g., a human)suffering from neurogenic inflammation in combination with a compositionof the invention include, but are not limited to, hydrocortisone,cortisone acetate, prednisone, prednisolone, methylprednisolone,dexamethasone, betamethasone, triamcinolone, beclometasone,fludrocortisone acetate, deoxycorticosterone acetate, aldosterone, andpharmaceutically acceptable salts thereof.

Narcotics that can be administered to a patient (e.g., a human)suffering from neurogenic inflammation in combination with a compositionof the invention include, but are not limited, to tramadol, hydrocodone,oxycodone, morphine, and pharmaceutically acceptable salts thereof.

Antiproliferative and immune modulatory agents that can be administeredto a patient (e.g., a human) suffering from neurogenic inflammation incombination with a composition of the invention include, but are notlimited to, alkylating agents, platinum agents, antimetabolites,topoisomerase inhibitors, dihydrofolate reductase inhibitors, antitumorantibiotics, antimitotic agents, aromatase inhibitors, thymidylatesynthase inhibitors, DNA antagonists, farnesyltransferase inhibitors,pump inhibitors, histone acetyltransferase inhibitors, metalloproteinaseinhibitors, ribonucleoside reductase inhibitors, TNF-alpha agonists,TNF-alpha antagonists or scavengers, interleukin 1 (IL-1) antagonists orscavengers, endothelin A receptor antagonists, retinoic acid receptoragonists, hormonal agents, antihormonal agents, photodynamic agents, andtyrosine kinase inhibitors.

The biologically active agents can be administered prior to, concurrentwith, or following administration of a composition of the invention,using any formulation, dosing, or administration known in the art thatis therapeutically effective.

Formulation of Compositions

The administration of the compounds of the invention may be by anysuitable means that results in the reduction of perceived pain sensationat the target region. The compounds of the invention may be contained inany appropriate amount in any suitable carrier substance, and aregenerally present in amounts totaling 1-99% by weight of the totalweight of the composition. The composition may be provided in a dosageform that is suitable for oral, parenteral (e.g., intravenous,intramuscular), rectal, cutaneous, subcutaneous, topical, transdermal,sublingual, nasal, vaginal, intrathecal, epidural, or ocularadministration, or by injection, inhalation, or direct contact with thenasal or oral mucosa.

Thus, the composition may be in the form of, e.g., tablets, capsules,pills, powders, granulates, suspensions, emulsions, solutions, gelsincluding hydrogels, pastes, ointments, creams, plasters, drenches,osmotic delivery devices, suppositories, enemas, injectables, implants,sprays, or aerosols. The compositions may be formulated according toconventional pharmaceutical practice (see, e.g., Remington: The Scienceand Practice of Pharmacy, 22nd edition, 2013, ed. L. V. Allen,Pharmaceutical Press, Philadelphia, and Encyclopedia of PharmaceuticalTechnology, 4^(th) Edition, ed. J. Swarbrick, 2013, CRC Press, NewYork).

Each compound may be formulated in a variety of ways that are known inthe art. For example, a compound of the invention and a biologicallyactive agent as defined herein may be formulated together or separately.Desirably, a compound of the invention and a biologically active agentare formulated together for their simultaneous or near simultaneousadministration. In another embodiment, two or more biologically activeagents may be formulated together with a compound of the invention, orseparately. Other examples include, but are not limited to, two or morecompounds of the invention formulated together, wherein the compoundsare formulated together with or without one or more biologically activeagents.

The individually or separately formulated agents can be packagedtogether as a kit. Non-limiting examples include but are not limited tokits that contain, e.g., two pills, a pill and a powder, a suppositoryand a liquid in a vial, two topical creams, etc. The kit can includeoptional components that aid in the administration of the unit dose topatients, such as vials for reconstituting powder forms, syringes forinjection, customized IV delivery systems, inhalers, etc. Additionally,the unit dose kit can contain instructions for preparation andadministration of the compositions.

The kit may be manufactured as a single use unit dose for one patient,multiple uses for a particular patient (at a constant dose or in whichthe individual compounds may vary in potency as therapy progresses); orthe kit may contain multiple doses suitable for administration tomultiple patients (“bulk packaging”). The kit components may beassembled in cartons, blister packs, bottles, tubes, and the like.

Controlled Release Formulations

Each compound of the invention, alone or in combination with one or moreof the biologically active agents as described herein, can be formulatedfor controlled release (e.g., sustained or measured) administration, asdescribed in U.S. Patent Application Publication Nos. 2003/0152637 and2005/0025765, each incorporated herein by reference. For example, acompound of the invention, alone or in combination with one or more ofthe biologically active agents as described herein, can be incorporatedinto a capsule or tablet that is administered to the patient.

Any pharmaceutically acceptable vehicle or formulation suitable forlocal application and/or injection into a site to be treated (e.g., apainful surgical incision, wound, or joint), that is able to provide asustained release of compound of the invention, alone or in combinationwith one or more of the biologically active agents as described herein,may be employed to provide for prolonged elimination or alleviation ofinflammation, as needed. Controlled release formulations known in theart include specially coated pellets, polymer formulations or matricesfor surgical insertion or as sustained release microparticles, e.g.,microspheres or microcapsules, for implantation, insertion, infusion orinjection, wherein the slow release of the active medicament is broughtabout through sustained or controlled diffusion out of the matrix and/orselective breakdown of the coating of the preparation or selectivebreakdown of a polymer matrix. Other formulations or vehicles forcontrolled, sustained or immediate delivery of an agent to a preferredlocalized site in a patient include, e.g., suspensions, emulsions, gels,liposomes and any other suitable art known delivery vehicle orformulation acceptable for subcutaneous or intramuscular administration.

A wide variety of biocompatible materials may be utilized as acontrolled release carrier to provide the controlled release of acompound of the invention, alone or in combination with one or morebiologically active agents, as described herein. Any pharmaceuticallyacceptable biocompatible polymer known to those skilled in the art maybe utilized. It is preferred that the biocompatible controlled releasematerial degrade in vivo within about one year, preferably within about3 months, more preferably within about two months. More preferably, thecontrolled release material will degrade significantly within one tothree months, with at least 50% of the material degrading into non-toxicresidues, which are removed by the body, and 100% of the compound of theinvention being released within a time period within about two weeks,preferably within about 2 days to about 7 days. A degradable controlledrelease material should preferably degrade by hydrolysis, either bysurface erosion or bulk erosion, so that release is not only sustainedbut also provides desirable release rates. However, the pharmacokineticrelease profile of these formulations may be first order, zero order,bi- or multi-phasic, to provide the desired reversible localanti-nociceptive effect over the desired time period.

Suitable biocompatible polymers can be utilized as the controlledrelease material. The polymeric material may comprise biocompatible,biodegradable polymers, and, in certain preferred embodiments, ispreferably a copolymer of lactic and glycolic acid. Preferred controlledrelease materials which are useful in the formulations of the inventioninclude the polyanhydrides, polyesters, co-polymers of lactic acid andglycolic acid (preferably wherein the weight ratio of lactic acid toglycolic acid is no more than 4:1 i.e., 80% or less lactic acid to 20%or more glycolic acid by weight) and polyorthoesters containing acatalyst or degradation enhancing compound, for example, containing atleast 1% by weight anhydride catalyst such as maleic anhydride. Examplesof polyesters include polylactic acid, polyglycolic acid and polylacticacid-polyglycolic acid copolymers. Other useful polymers include proteinpolymers such as collagen, gelatin, fibrin and fibrinogen andpolysaccharides such as hyaluronic acid.

The polymeric material may be prepared by any method known to thoseskilled in the art. For example, where the polymeric material iscomprised of a copolymer of lactic and glycolic acid, this copolymer maybe prepared by the procedure set forth in U.S. Pat. No. 4,293,539,incorporated herein by reference. Alternatively, copolymers of lacticand glycolic acid may be prepared by any other procedure known to thoseskilled in the art. Other useful polymers include polylactides,polyglycolides, polyanhydrides, polyorthoesters, polycaprolactones,polyphosphazenes, polyphosphoesters, polysaccharides, proteinaceouspolymers, soluble derivatives of polysaccharides, soluble derivatives ofproteinaceous polymers, polypeptides, polyesters, and polyorthoesters ormixtures or blends of any of these.

Pharmaceutically acceptable polyanhydrides which are useful in thepresent invention have a water-labile anhydride linkage. The rate ofdrug release can be controlled by the particular polyanhydride polymerutilized and its molecular weight. The polysaccharides may bepoly-1,4-glucans, e.g., starch glycogen, amylose, amylopectin, andmixtures thereof. The biodegradable hydrophilic or hydrophobic polymermay be a water-soluble derivative of a poly-1,4-glucan, includinghydrolyzed amylopectin, derivatives of hydrolyzed amylopectin such ashydroxyethyl starch (HES), hydroxyethyl amylose, dialdehyde starch, andthe like. The polyanhydride polymer may be branched or linear.

Examples of polymers which are useful in the present invention include(in addition to homopolymers and copolymers of poly(lactic acid) and/orpoly(glycolic acid)) poly[bis(p-carboxyphenoxy) propane anhydride](PCPP), poly[bis(p-carboxy)methane anhydride](PCPM), polyanhydrides ofoligomerized unsaturated aliphatic acids, polyanhydride polymersprepared from amino acids which are modified to include an additionalcarboxylic acid, aromatic polyanhydride compositions, and co-polymers ofpolyanhydrides with other substances, such as fatty acid terminatedpolyanhydrides, e.g., polyanhydrides polymerized from monomers of dimersand/or trimers of unsaturated fatty acids or unsaturated aliphaticacids. Polyanhydrides may be prepared in accordance with the methods setforth in U.S. Pat. No. 4,757,128, incorporated herein by reference.Polyorthoester polymers may be prepared, e.g., as set forth in U.S. Pat.No. 4,070,347, incorporated herein by reference. Polyphosphoesters maybe prepared and used as set forth in U.S. Pat. Nos. 6,008,318,6,153,212, 5,952,451, 6,051,576, 6,103,255, 5,176,907 and 5,194,581,each of which is incorporated herein by reference.

Proteinaceous polymers may also be used. Proteinaceous polymers andtheir soluble derivatives include gelation biodegradable syntheticpolypeptides, elastin, alkylated collagen, alkylated elastin, and thelike. Biodegradable synthetic polypeptides includepoly-(N-hydroxyalkyl)-L-asparagine, poly-(N-hydroxyalkyl)-L-glutamine,copolymers of N-hydroxyalkyl-L-asparagine and N-hydroxyalkyl-L-glutaminewith other amino acids. Suggested amino acids include L-alanine,L-lysine, L-phenylalanine, L-valine, L-tyrosine, and the like.

In additional embodiments, the controlled release material, which ineffect acts as a carrier for a compound of the invention, alone or incombination with one or more biologically active agents as describedherein, can further include a bioadhesive polymer such as pectins(polygalacturonic acid), mucopolysaccharides (hyaluronic acid, mucin) ornon-toxic lectins or the polymer itself may be bioadhesive, e.g.,polyanhydride or polysaccharides such as chitosan.

In embodiments where the biodegradable polymer comprises a gel, one suchuseful polymer is a thermally gelling polymer, e.g., polyethylene oxide,polypropylene oxide (PEO-PPO) block copolymer such as Pluronic™ F127from BASF Wyandotte. In such cases, the local anesthetic formulation maybe injected via syringe as a free-flowing liquid, which gels rapidlyabove 30° C. (e.g., when injected into a patient). The gel system thenreleases a steady dose of a compound of the invention, alone or incombination with one or more biologically active agents as describedherein, at the site of administration.

Dosage Forms for Oral Use

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystallinecellulose, starches including potato starch, calcium carbonate, sodiumchloride, lactose, calcium phosphate, calcium sulfate, or sodiumphosphate); granulating and disintegrating agents (e.g., cellulosederivatives including microcrystalline cellulose, starches includingpotato starch, croscarmellose sodium, alginates, or alginic acid);binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid,sodium alginate, gelatin, starch, pregelatinized starch,microcrystalline cellulose, magnesium aluminum silicate,carboxymethylcellulose sodium, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethyleneglycol); and lubricating agents, glidants, and antiadhesives (e.g.,magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenatedvegetable oils, or talc). Other pharmaceutically acceptable excipientscan be colorants, flavoring agents, plasticizers, humectants, bufferingagents, taste masking agents (such as hydroxypropyl methylcellulose,hydroxypropyl cellulose), and the like.

One or more compounds of the invention and one or more biologicallyactive agents, as defined herein, may be mixed together in a tablet,capsule, or other vehicle, or may be partitioned. In one example, acompound of the invention is contained on the inside of the tablet, andthe biologically active agent is on the outside of the tablet, such thata substantial portion of the biologically active agent is released priorto the release of the compound of the invention.

Formulations for oral use may also be provided as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent (e.g., potato starch, lactose, microcrystallinecellulose, calcium carbonate, calcium phosphate or kaolin), or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example, peanut oil, liquid paraffin, or olive oil.Powders, granulates, and pellets may be prepared using the ingredientsmentioned above under tablets and capsules in a conventional mannerusing, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.

Formulations for oral administration to the mouth may also be providedas a mouthwash, an oral spray, oral rinse solution, oral ointment, ororal gel.

Dissolution or diffusion controlled release can be achieved byappropriate coating of a tablet, capsule, pellet, or granulateformulation of compounds, or by incorporating the compound into anappropriate matrix. A controlled release coating may include one or moreof the coating substances mentioned above and/or, e.g., shellac,beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glycerylmonostearate, glyceryl distearate, glycerol palmitostearate,ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetatebutyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone,polyethylene, polymethacrylate, methylmethacrylate,2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol,ethylene glycol methacrylate, and/or polyethylene glycols. In acontrolled release matrix formulation, the matrix material may alsoinclude, e.g., hydrated methylcellulose, carnauba wax and stearylalcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

The liquid forms in which the compounds and compositions of the presentinvention can be incorporated for administration orally include aqueoussolutions, suitably flavored syrups, aqueous or oil suspensions, andflavored emulsions with edible oils such as cottonseed oil, sesame oil,coconut oil, or peanut oil, as well as elixirs and similarpharmaceutical vehicles.

Generally, when administered to a human, the oral dosage of any of thecompounds of the combination of the invention will depend on the natureof the compound, and can readily be determined by one skilled in theart. Typically, such dosage is normally about 0.001 mg to 2000 mg perday, desirably about 1 mg to 1000 mg per day, and more desirably about 5mg to 500 mg per day. Dosages up to 200 mg per day may be necessary.

Administration of each drug in a combination therapy, as describedherein, can, independently, be one to four times daily for one day toone year, and may even be for the life of the patient. Chronic,long-term administration will be indicated in many cases.

Parenteral Formulations Formulations suitable for parenteraladministration (e.g., by injection), include aqueous or non-aqueous,isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions),in which the compound is dissolved, suspended, or otherwise provided(e.g., in a liposome or other microparticulate). Such liquids mayadditional contain other pharmaceutically acceptable ingredients, suchas anti-oxidants, buffers, preservatives, stabilisers, bacteriostats,suspending agents, thickening agents, and solutes which render theformulation isotonic with the blood (or other relevant bodily fluid) ofthe intended recipient. Examples of excipients include, for example,water, alcohols, polyols, glycerol, vegetable oils, and the like.Examples of suitable isotonic carriers for use in such formulationsinclude Sodium Chloride Injection, Ringer's Solution, or LactatedRinger's Injection. Typically, the concentration of the compound in theliquid is from about 1 ng/ml to about 10 μg/ml, for example from about10 ng/ml to about 1 μg/ml. The formulations may be presented inunit-dose or multi-dose sealed containers, for example, ampoules andvials, and may be stored in a freeze-dried (lyophilised) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders,granules, and tablets.

Topical Formulations

The compositions of the invention, alone or in combination with one ormore of the biologically active agents described herein, can also beadapted for topical use with a topical vehicle containing from between0.0001% and 25% (w/w) or more of active ingredient(s).

In a preferred combination, the active ingredients are preferably eachfrom between 0.0001% to 10% (w/w), more preferably from between 0.0005%to 4% (w/w) active agent. The topical formulation, including but notlimited to a cream, gel, or ointment, can be applied one to four timesdaily, or as needed. Performing the methods described herein, thetopical vehicle containing the composition of the invention, or acombination therapy containing a composition of the invention ispreferably applied to the site of inflammation on the patient. Forexample, a cream may be applied to the hands of a patient suffering fromarthritic fingers.

The compositions can be formulated using any dermatologically acceptablecarrier. Exemplary carriers include a solid carrier, such as alumina,clay, microcrystalline cellulose, silica, or talc; and/or a liquidcarrier, such as an alcohol, a glycol, or a water-alcohol/glycol blend.The therapeutic agents may also be administered in liposomalformulations that allow therapeutic agents to enter the skin. Suchliposomal formulations are described in U.S. Pat. Nos. 5,169,637;5,000,958; 5,049,388; 4,975,282; 5,194,266; 5,023,087; 5,688,525;5,874,104; 5,409,704; 5,552,155; 5,356,633; 5,032,582; 4,994,213;8,822,537, and PCT Publication No. WO 96/40061. Examples of otherappropriate vehicles are described in U.S. Pat. Nos. 4,877,805,8,822,537, and EP Publication No. 0586106A1. Suitable vehicles of theinvention may also include mineral oil, petrolatum, polydecene, stearicacid, isopropyl myristate, polyoxyl 40 stearate, stearyl alcohol, orvegetable oil.

The composition can further include a skin penetrating enhancer, such asthose described in “Percutaneous Penetration enhancers”, (eds. Smith E Wand Maibach H I. CRC Press 1995). Exemplary skin penetrating enhancersinclude alkyl (N,N-disubstituted amino alkanoate) esters, such asdodecyl 2-(N,N dimethylamino) propionate (DDAIP), which is described inpatents U.S. Pat. Nos. 6,083,996 and 6,118,020, which are bothincorporated herein by reference; a water-dispersible acid polymer, suchas a polyacrylic acid polymer, a carbomer (e.g., Carbopol™ or Carbopol940P™, available from B. F. Goodrich Company (Akron, Ohio)), copolymersof polyacrylic acid (e.g., Pemulen™ from B. F. Goodrich Company orPolycarbophil™ from A. H. Robbins, Richmond, Va.; a polysaccharide gum,such as agar gum, alginate, carrageenan gum, ghatti gum, karaya gum,kadaya gum, rhamsan gum, xanthan gum, and galactomannan gum (e.g., guargum, carob gum, and locust bean gum), as well as other gums known in theart (see for instance, Industrial Gums: Polysaccharides & TheirDerivatives, Whistler R. L., BeMiller J. N. (eds.), 3rd Ed. AcademicPress (1992) and Davidson, R. L., Handbook of Water-Soluble Gums &Resins, McGraw-Hill, Inc., N.Y. (1980)); or combinations thereof.

Other suitable polymeric skin penetrating enhancers are cellulosederivatives, such as ethyl cellulose, methyl cellulose, hydroxypropylcellulose. Additionally, known transdermal penetrating enhancers canalso be added, if desired. Illustrative are dimethyl sulfoxide (DMSO)and dimethyl acetamide (DMA), 2-pyrrolidone, N,N-diethyl-m-toluamide(DEET), 1-dodecylazacycloheptane-2-one (Azone™, a registered trademarkof Nelson Research), N,N-dimethylformamide, N-methyl-2-pyrrolidone,calcium thioglycolate and other enhancers such as dioxolanes, cyclicketones, and their derivatives and so on.

Also illustrative are a group of biodegradable absorption enhancerswhich are alkyl N,N-2-(disubstituted amino) alkanoates as described inU.S. Pat. Nos. 4,980,378 and 5,082,866, which are both incorporatedherein by reference, including: tetradecyl (N,N-dimethylamino) acetate,dodecyl (N,N-dimethylamino) acetate, decyl (N,N-dimethylamino) acetate,octyl (N,N-dimethylamino) acetate, and dodecyl (N,N-diethylamino)acetate.

Particularly preferred skin penetrating enhancers include isopropylmyristate; isopropyl palmitate; dimethyl sulfoxide; decyl methylsulfoxide; dimethylalanine amide of a medium chain fatty acid; dodecyl2-(N,N-dimethylamino) propionate or salts thereof, such as its organic(e.g., hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric acidaddition salts) and inorganic salts (e.g., acetic, benzoic, salicylic,glycolic, succinic, nicotinic, tartaric, maleic, malic, pamoic,methanesulfonic, cyclohexanesulfamic, picric, and lactic acid additionsalts), as described in U.S. Pat. No. 6,118,020; and alkyl2-(N,N-disubstituted amino)-alkanoates, as described in U.S. Pat. Nos.4,980,378 and 5,082,866.

The skin penetrating enhancer in this composition by weight would be inthe range of 0.5% to 10% (w/w). The most preferred range would bebetween 1.0% and 5% (w/w). In another embodiment, the skin penetratingenhancer comprises between 0.5%-1%, 1%-2%, 2%-3%, 3%-4%, or 4%-5%, (w/w)of the composition.

The compositions can be provided in any useful form. For example, thecompositions of the invention may be formulated as solutions, emulsions(including microemulsions), suspensions, creams, ointments, foams,lotions, gels, powders, or other typical solid, semi-solid, or liquidcompositions (e.g., topical sprays) used for application to the skin orother tissues where the compositions may be used. Such compositions maycontain other ingredients typically used in such products, such ascolorants, fragrances, thickeners (e.g., xanthan gum, a fatty acid, afatty acid salt or ester, a fatty alcohol, a modified cellulose, amodified mineral material, Krisgel 100™, or a synthetic polymer),antimicrobials, solvents, surfactants, detergents, gelling agents,antioxidants, fillers, dyestuffs, viscosity-controlling agents,preservatives, humectants, emollients (e.g., natural or synthetic oils,hydrocarbon oils, waxes, or silicones), hydration agents, chelatingagents, demulcents, solubilizing excipients, adjuvants, dispersants,skin penetrating enhancers, plasticizing agents, preservatives,stabilizers, demulsifiers, wetting agents, sunscreens, emulsifiers,moisturizers, astringents, deodorants, and optionally includinganesthetics, anti-itch actives, botanical extracts, conditioning agents,darkening or lightening agents, glitter, humectants, mica, minerals,polyphenols, silicones or derivatives thereof, sunblocks, vitamins, andphytomedicinals.

The compositions can also include other like ingredients to provideadditional benefits and improve the feel and/or appearance of thetopical formulation. Specific classes of additives commonly use in theseformulations include: isopropyl myristate, sorbic acid NF powder,polyethylene glycol, phosphatidylcholine (including mixtures ofphosphatidylcholine, such as phospholipon G), Krisgel 100™ distilledwater, sodium hydroxide, decyl methyl sulfoxide (as a skin penetratingenhancer), menthol crystals, lavender oil, butylated hydroxytoluene,ethyl diglycol reagent, and 95% percent (190 proof) ethanol.

Formulations for Ophthalmic Administration

The compounds of the invention can also be formulated with anophthalmically acceptable carrier in sufficient concentration so as todeliver an effective amount of the active compound or compounds to theoptic nerve site of the eye. Preferably, the ophthalmic, therapeuticsolutions contain one or more of the active compounds in a concentrationrange of approximately 0.0001% to approximately 5% (weight by volume)and more preferably approximately 0.0005% to approximately 0.1% (weightby volume).

An ophthalmically acceptable carrier does not cause significantirritation to the eye and does not abrogate the pharmacological activityand properties of the charged sodium channel blockers.

Ophthalmically acceptable carriers are generally sterile, essentiallyfree of foreign particles, and generally have a pH in the range of 5-8.Preferably, the pH is as close to the pH of tear fluid (7.4) aspossible. Ophthalmically acceptable carriers are, for example, sterileisotonic solutions such as isotonic sodium chloride or boric acidsolutions. Such carriers are typically aqueous solutions contain sodiumchloride or boric acid. Also useful are phosphate buffered saline (PBS)solutions.

Various preservatives may be used in the ophthalmic preparation.Preferred preservatives include, but are not limited to, benzalkoniumpotassium, chlorobutanol, thimerosal, phenylmercuric acetate, andphenylmercuric nitrate. Likewise, various preferred vehicles may be usedin such ophthalmic preparation. These vehicles include, but are notlimited to, polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose,poloxamers, carboxymethyl cellulose and hydroxyethyl cellulose.

Tonicity adjustors may be added as needed or convenient. They include,but are not limited to, salts, particularly sodium chloride, potassiumchloride, etc., mannitol and glycerin, or any other suitableophthalmically acceptable tonicity adjustor.

Various buffers and means for adjusting pH may be used so long as theresulting preparation is ophthalmically acceptable. Accordingly, buffersinclude but are not limited to, acetate buffers, citrate buffers,phosphate buffers, and borate buffers. Acids or bases may be used toadjust the pH of these formulations as needed. Ophthalmically acceptableantioxidants can also be include. Antioxidants include but are notlimited to sodium metabisulfite, sodium thiosulfate, acetylcysteine,butylated hydroxyanisole, and butylated hydroxytoluene.

Formulations for Nasal and Inhalation Administration

The pharmaceutical compositions of the invention can be formulated fornasal or intranasal administration. Formulations suitable for nasaladministration, when the carrier is a solid, include a coarse powderhaving a particle size, for example, in the range of approximately 20 to500 microns which is administered by rapid inhalation through the nasalpassage. When the carrier is a liquid, for example, a nasal spray or asnasal drops, one or more of the formulations can be admixed in anaqueous or oily solution, and inhaled or sprayed into the nasal passage.

For administration by inhalation, the active ingredient can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit can be determined byproviding a valve to deliver a metered amount, Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator can beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

Dry powder compositions for topical delivery to the lung by inhalationmay, for example, be presented in capsules and cartridges of, forexample, gelatin or blisters of, for example, laminated aluminum foil,for use in an inhaler or insufflator. Powder blend formulationsgenerally contain a powder mix for inhalation of the compound of theinvention and a suitable powder base (carrier/diluent/excipientsubstance) such as mono-, di or ploy-saccharides (e.g. lactose orstarch). Use of lactose is preferred. In one embodiment, each capsule orcartridge may contain between about 2 ug to about 100 mg of the compoundof formula (I) optionally in combination with another therapeuticallyactive ingredient. In a preferred embodiment, each capsule or cartridgemay contain between about 10 ug to about 50 mg of the compound offormula (I) optionally in combination with another therapeuticallyactive ingredient. In another embodiment, each capsule or cartridge maycontain between about 20 ug to about 10 mg of the compound of formula(I) optionally in combination with another therapeutically activeingredient. Alternatively, the compound of the invention may bedelivered without excipients.

Suitably, the packaging/medicament dispenser is of a type selected fromthe group consisting of a reservoir dry powder inhaler (RDPI), a unitdose dry powder inhaler (e.g., a capsule or blister pack inhaler), amulti-dose dry powder inhaler (MDPI), and a metered dose inhaler (MDI).

Solutions or suspensions for use in a pressurized container, pump,spray, atomizer, or nebulizer can be formulated to contain an aqueousmedium, ethanol, aqueous ethanol, or a suitable alternative agent fordispersing, solubilizing, or extending release of the activeingredient(s); a propellant as solvent; and/or a surfactant, such assorbitan trioleate, oleic acid, or an oligolactic acid.

Compositions formulated for nasal or inhalation administration mayinclude one or more taste-masking agents such as flavoring agents,sweeteners, and other strategies, such as sucrose, dextrose, andlactose, carboxylic acids, menthol, amino acids or amino acidderivatives such as arginine, lysine, and monosodium glutamate, and/orsynthetic flavor oils and flavoring aromatics and/or natural oils,extracts from plants, leaves, flowers, fruits, etc. and combinationsthereof. These may include cinnamon oils, oil of wintergreen, peppermintoils, clover oil, bay oil, anise oil, eucalyptus, vanilla, citrus oilsuch as lemon oil, orange oil, grape and grapefruit oil, fruit essencesincluding apple, peach, pear, strawberry, raspberry, cherry, plum,pineapple, apricot, etc. Additional sweeteners include sucrose,dextrose, aspartame, acesulfame-K, sucralose and saccharin, organicacids (by non-limiting example citric acid and aspartic acid). Suchflavors may be present at from about 0.05 to about 4 percent by weight,and may be present at lower or higher amounts as a factor of one or moreof potency of the effect on flavor, solubility of the flavorant, effectsof the flavorant on solubility or other physicochemical orpharmacokinetic properties of other formulation components, or otherfactors.

Indications

The compounds, compositions, methods, and kits of the invention can beused to treat pain, cough or itch associated with any of a number ofconditions, including trigeminal trophic syndrome, erythromelalgia, backand neck pain, lower back pain, cancer pain, gynecological and laborpain, abdominal wall pain, chronic abdominal wall pain, fibromyalgia,allergic rhinitis, arthritis, rheumatoid arthritis, osteoarthritis,rheumatological pains, orthopedic pains, acute and post herpeticneuralgia and other neuropathic pains (including peripheral neuropathy),sickle cell crises, muscle pain, vulvodynia, rectal pain, Levator anisyndrome, proctalgia fugax, peri-anal pain, hemorrhoid pain, stomachpain, ulcers, inflammatory bowel disease, irritable bowel disease,irritable bowel syndrome, oral mucositis, esophagitis, interstitialcystitis, urethritis and other urological pains, dental pain, burn pain,headaches, ophthalmic irritation, conjunctivitis (e.g., allergicconjunctivitis), eye redness, dry eye, dry eye syndrome (chronic ocularpain), complex regional pain syndrome, acute postoperative pain,postoperative pain, post-surgical ocular pain, and procedural pain(i.e., pain associated with injections, draining an abscess, surgery,dental procedures, ophthalmic procedures, arthroscopies and use of othermedical instrumentation, cosmetic surgical procedures, dermatologicalprocedures, setting fractures, biopsies, and the like).

Since a subclass of nociceptors mediate itch sensation, the compounds,compositions, methods, and kits of the invention can also be used totreat itch in patients with conditions like pruritus (including, but notlimited to, brachioradial, chronic idiopathic, genital/anal, notalgiaparesthetica, and scalp), allergic dermatitis, atopic dermatitis,contact dermatitis, poison ivy, infections, parasites, insect bites,pregnancy, metabolic disorders, liver or renal failure, drug reactions,allergic reactions, eczema, hand eczema, genital and anal itch,hemorrhoid itch, and cancer.

Since a subclass of nociceptors can initiate aberrant cough reflexes,the compounds, compositions, methods, and kits of the invention can alsobe used to treat cough in patients with conditions like asthma, COPD,asthma-COPD overlap syndrome (ACOS), interstitial pulmonary fibrosis(IPF), idiopathic pulmonary fibrosis, post viral cough, post-infectioncough, chronic idiopathic cough and lung cancer.

The compounds, compositions, methods, and kits of the invention can alsobe used to treat neurogenic inflammation and neurogenic inflammatorydisorders. Inflammation is a complex set of responses to harmful stimulithat results in localized redness, swelling, and pain. Inflammation canbe innate or adaptive, the latter driven by antigens and is mediated byimmune cells (immune-mediated inflammation). Neurogenic inflammationresults from the efferent functions of pain-sensing neurons(nociceptors), wherein neuropeptides and other chemicals that arepro-inflammatory mediators are released from the peripheral terminals ofthe nociceptors when they are activated. This release process ismediated by calcium influx and exocytosis of peptide containingvesicles, and the pro-inflammatory neuropeptides include substance P,neurokinin A and B (collectively known as tachykinins), calcitoningene-related peptide (CGRP), and vasoactive intestinal polypeptide(VIP).

The release of peripheral terminal chemicals stimulate a variety ofinflammatory responses. First, the release of substance P can result inan increase in capillary permeability such that plasma proteins leakfrom the intravascular compartment into the extracellular space (plasmaextravasation), causing edema. This can be detected as a wheal (a firm,elevated swelling of the skin) which is one component of a triad ofinflammatory responses—wheal, red spot, and flare—known as the Lewistriple response. Second, the release of CGRP causes vasodilation,leading to increased blood flow. This can be detected as a flare, whichis another component of the Lewis triple response.

Substance P also has a pro-inflammatory action on immune cells (e.g.macrophages, T-cells, mast cells, and dendritic cells) via theirneurokinin-1 (NK1) receptor. This effect has been documented in allergicrhinitis, gastritis, and colitis, and represents an interface betweenthe neurogenic and immune-mediated components of inflammation. SubstanceP released from one nociceptor may also act on NK1 receptors onneighboring nociceptors to sensitize or activate them, causing a spreadof activation and afferent/efferent function. These efferent functionsof nociceptors can be triggered by: 1) Direct activation of a nociceptorterminal by a peripheral adequate stimulus applied to the terminal (e.g.a pinch); 2) Indirect antidromic activation of a non-stimulatednociceptor terminal by the axon reflex, wherein action potential inputfrom one terminal of a nociceptor, upon reaching a converging axonalbranch point in the periphery, results in an action potential travelingfrom the branch point down to the peripheral terminal of anon-stimulated terminal; and 3) Activation as a result of activity innociceptor central terminals in the CNS traveling to the periphery(e.g., primary afferent depolarization of central terminals produced byGABA can be sufficient to initiate action potentials traveling the“wrong way”).

Genomic analysis of lung resident ILC2 cells has revealed expression ofreceptors for several neuropeptides released by sensory neurons,including SP, CGRP and VIP, providing an opportunity for nociceptors todirectly communicate with these cells. In particular, VIP is found to beexpressed in NaV1.8+ nodose ganglion neurons, including lung afferentsin OVA-exposed mice. Cultured nodose ganglion neurons stimulated withcapsaicin or IL5 also released VIP while BALF from OVA-exposed micecontained elevated VIP compared to vehicle-challenged mice (Talbot etal., Neuron. 2015 Jul. 15; 87(2): 341-354). These data indicate that VIPis released in the inflamed lung and can be blocked by silencing neuronswith charged sodium channel blockers of the present invention. Inaddition, when CD4+ T cells cultured under T_(H)2 skewing conditionswere exposed to recombinant mouse VIP, the transcript levels of IL-13and IL-5 increased, suggesting that VIP contributes to the competence ofT_(H)2 cells to transcribe these type II regulatory cytokines.

Immune mediator release from immune cells can also activate nociceptors.Mast cells are found close to primary nociceptive neurons and contributeto nociceptor sensitization in a number of contexts. Injection of thesecretagogue compound 48/80 promotes degranulation of mast cells in thedura and leads to excitation of meningeal nociceptors. Mast celldegranulation also contributes to the rapid onset of nerve growthfactor-induced thermal hyperalgesia. Macrophages contribute tonociceptor sensitization by releasing several soluble mediators.Expression of the chemokine macrophage inflammatory protein-la (MIP-la)and its receptors CCR1 and CCR5 is increased in macrophages and Schwanncells after partial ligation of the sciatic nerve and contributes to thedevelopment of neuropathic pain. Lymphocytes contribute to thesensitization of peripheral nociceptors. T cells infiltrate the sciaticnerve and dorsal root ganglion (DRG) after nerve injury. Hyperalgesiaand allodynia induced by nerve injury are markedly attenuated orabrogated in rodents lacking T cells and the immunosuppressant rapamycinattenuates neuropathic pain in rats, partly owing to an effect on Tcells. Among the subsets of T cells, type 1 and 2 helper T cells (T_(H)1and T_(H)2 cells) have been shown to have different roles in neuropathicpain. T_(H)1 cells facilitate neuropathic pain behavior by releasingproinflammatory cytokines (IL-2 and interferon-γ (IFNγ)), whereas T_(H)2cells inhibit it by releasing anti-inflammatory cytokines (IL-4, IL-10and IL-13). The complement system also has a role in inflammatoryhyperalgesia and neuropathic pain. C5a, an anaphylatoxin, is animportant effector of the complement cascade and upon binding to C5aR1receptors on neutrophils it becomes a potent neutrophil attractant (Ren& Dubner, Nat. Med. 16:1267-1276 (2010)).

Bacterial infections have been shown to directly activate nociceptors,and that the immune response mediated through TLR2, MyD88, T cells, Bcells, and neutrophils and monocytes is not necessary for Staphylococcusaureus-induced pain in mice (Chiu et al., Nature 501:52-57 (2013)).Mechanical and thermal hyperalgesia in mice is correlated with livebacterial load rather than tissue swelling or immune activation.Bacteria induce calcium flux and action potentials in nociceptorneurons, in part via bacterial N-formylated peptides and thepore-forming toxin α-haemolysin, through distinct mechanisms. Specificablation of Nav1.8-lineage neurons, which include nociceptors, abrogatedpain during bacterial infection, but concurrently increased local immuneinfiltration and lymphadenopathy of the draining lymph node. Thus,bacterial pathogens produce pain by directly activating sensory neuronsthat modulate inflammation, an unsuspected role for the nervous systemin host-pathogen interactions. Data from Talbot et al., (Neuron. 2015Jul. 15; 87(2): 341-354.) have also suggested that nociceptors areactivated during exposure to allergens in sensitized animals.

In certain disorders, neurogenic inflammation contributes to theperipheral inflammation elicited by tissue injury, autoimmune disease,infection, and exposure to irritants in soft tissue, skin, therespiratory system, joints, the urogenital and GI tract, the liver, andthe brain. Neurogenic inflammatory disorders include, but are notlimited to, allergic inflammation, inflammatory bowel disease,interstitial cystitis, atopic dermatitis, asthma, conjunctivitis,arthritis, colitis, contact dermatitis, diabetes, eczema, cystitis,gastritis, migraine headache, psoriasis, rhinitis, rosacea, sunburn,pancreatitis, chronic cough, chronic rhinosinusistis, traumatic braininjury, polymicrobial sepsis, tendinopathies, chronic urticaria,rheumatic disease, acute lung injury, exposure to irritants, inhalationof irritants, pollutants, or chemical warfare agents, as describedherein.

Assessment of Pain, Cough, Itch, and Neurogenic Inflammation

In order to measure the efficacy of any of the compounds, compositions,methods, and kits of the invention in the treatment of pain associatedwith musculoskeletal, immunoinflammatory and neuropathic disorders, ameasurement index may be used. Indices that are useful include a visualanalog scale (VAS), a Likert scale, categorical pain scales,descriptors, the Lequesne index, the WOMAC index, and the AUSCAN index,each of which is well known in the art. Such indices may be used tomeasure pain, itch, function, stiffness, or other variables.

A visual analog scale (VAS) provides a measure of a one-dimensionalquantity. A VAS generally utilizes a representation of distance, such asa picture of a line with hash marks drawn at regular distance intervals,e.g., ten 1-cm intervals. For example, a patient can be asked to rank asensation of pain or itch by choosing the spot on the line that bestcorresponds to the sensation of pain or itch, where one end of the linecorresponds to “no pain” (score of 0 cm) or “no itch” and the other endof the line corresponds to “unbearable pain” or “unbearable itch” (scoreof 10 cm). This procedure provides a simple and rapid approach toobtaining quantitative information about how the patient is experiencingpain or itch. VAS scales and their use are described, e.g., in U.S. Pat.Nos. 6,709,406 and 6,432,937.

A Likert scale similarly provides a measure of a one-dimensionalquantity. Generally, a Likert scale has discrete integer values rangingfrom a low value (e.g., 0, meaning no pain) to a high value (e.g., 7,meaning extreme pain). A patient experiencing pain is asked to choose anumber between the low value and the high value to represent the degreeof pain experienced. Likert scales and their use are described, e.g., inU.S. Pat. Nos. 6,623,040 and 6,766,319.

The Lequesne index and the Western Ontario and McMaster Universities(WOMAC) osteoarthritis index assess pain, function, and stiffness in theknee and hip of OA patients using self-administered questionnaires. Bothknee and hip are encompassed by the WOMAC, whereas there is one Lequesnequestionnaire for the knee and a separate one for the hip. Thesequestionnaires are useful because they contain more information contentin comparison with VAS or Likert. Both the WOMAC index and the Lequesneindex questionnaires have been extensively validated in OA, including insurgical settings (e.g., knee and hip arthroplasty). Their metriccharacteristics do not differ significantly.

The AUSCAN (Australian-Canadian hand arthritis) index employs a valid,reliable, and responsive patient self-reported questionnaire. In oneinstance, this questionnaire contains 15 questions within threedimensions (Pain, 5 questions; Stiffness, 1 question; and Physicalfunction, 9 questions). An AUSCAN index may utilize, e.g., a Likert or aVAS scale.

Indices that are useful in the methods, compositions, and kits of theinvention for the measurement of pain include the Pain Descriptor Scale(PDS), the Visual Analog Scale (VAS), the Verbal Descriptor Scales(VDS), the Numeric Pain Intensity Scale (NPIS), the Neuropathic PainScale (NPS), the Neuropathic Pain Symptom Inventory (NPSI), the PresentPain Inventory (PPI), the Geriatric Pain Measure (GPM), the McGill PainQuestionnaire (MPQ), mean pain intensity (Descriptor DifferentialScale), numeric pain scale (NPS) global evaluation score (GES) theShort-Form McGill Pain Questionnaire, the Minnesota MultiphasicPersonality Inventory, the Pain Profile and Multidimensional PainInventory, the Child Heath Questionnaire, and the Child AssessmentQuestionnaire.

Itch can be measured by subjective measures (VAS, Lickert, descriptors).Another approach is to measure scratch which is an objective correlateof itch using a vibration transducer or movement-sensitive meters.

Cough can be measured by standard questionnaires like the LeicesterCough Questionnaire as well as validated objective instruments tomeasure cough frequency (e.g. VitaloJAK).

EXAMPLES

The following examples are intended to illustrate the invention, and arenot intended to limit it.

Example 1—Compound Syntheses General Abbreviation Definitions

ACN acetonitrileaq. aqueous° C. degrees Celsiusδ chemical shift (ppm)DCM dichloromethaneDMSO dimethyl sulfoxideESI electrospray ionizationEt₂O diethyl etherEtOAc ethyl acetateh hourMeOH methanolmHz megahertzmin minml milliliterMS mass spectrometrym/z mass to charge ratioNAMR nuclear magnetic resonancePet ether petroleum etherRT room temperatureTLC thin layer chromatographyUV ultraviolet light

Synthesis of 1-(2-((2, 6-dimethylphenyl) amino)-2-oxoethyl)pyridin-1-ium bromide

Synthesis of Intermediate 2-bromo-N-(2,6-dimethylphenyl)acetamide

To a suspension of 2, 6-dimethylaniline (30.5 mL, 247.56 mmol, 1.0 eq)in water (300 mL) was added bromoacetyl bromide (23.8 mL, 272.31 mmol,1.1 eq) at 10° C. The reaction mixture was maintained at pH 9-10 with15% Na₂CO₃ (aq.) solution for 1 hour as the progress of the reaction wasmonitored by TLC (mobile phase: 30% EtOAc in hexane, visualization byUV). The reaction mixture was extracted with EtOAc (2×600 mL) and thecombined organic extracts were washed with brine (200 mL), dried overNa₂SO₄, filtered and concentrated under reduced pressure. The resultingcrude product was triturated with Et₂O (2×200 mL) to afford2-bromo-N-(2, 6-dimethylphenyl) acetamide (21 g) as a white solid. MS(ESI): m/z 244.02 [M+2]⁺. ¹H NMR (400 MHz, CDCl₃) δ 7.75 (br s, 1H),7.20-7.02 (m, 3H), 4.07 (s, 2H), 2.24 (s, 6H).

Synthesis of 1-(2-((2, 6-dimethylphenyl) amino)-2-oxoethyl)pyridin-1-ium bromide

To a stirred solution of 2-bromo-N-(2,6-dimethylphenyl) acetamide (0.25g, 1.03 mmol, 1.0 eq) in EtOAc (10 mL) was added pyridine (0.166 mL,2.06 mmol, 2.0 eq) and the resulting mixture was stirred at 80° C. in asealed tube while consumption of starting material was monitored by TLC(mobile phase: 10% MeOH in DCM, visualization by UV). After 16 hours,the crude solution was cooled to room temperature and the resultingprecipitate was collected by filtration, washed with EtOAc (2×10 mL) anddried under vacuum to affordN-(2,6-dimethylphenyl)-2-(pyridinium-1-yl)acetamide bromide (0.1 g) as awhite solid. MS (ESI): m/z 241.1 [M]+. 1H NMR (400 MHz, DMSO-d6) δ 10.01(s, 1H), 9.08 (d, J=5.5 Hz, 2H), 8.69 (t, J=7.8 Hz, 1H), 8.22 (dd,J=6.8, 7.5 Hz, 2H), 7.16-6.99 (m, 3H), 5.75 (s, 2H), 2.20 (s, 6H).

Synthesis of3-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-methyl-1H-imidazol-3-iumbromide

Synthesis of 3-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-methyl-1H-imidazol-3-ium bromide

Synthesis of intermediate 2-bromo-N-(2,6-dimethylphenyl) acetamide wasas described above for Compound 1. To a stirred solution of2-bromo-N-(2,6-dimethylphenyl) acetamide (0.2 g, 0.8 mmol, 1.0 eq) inEtOAc (3 mL) was added 1-methyl-1H-imidazole (0.072 mL, 0.88 mmol, 1.1eq) and the resulting mixture was stirred at 80° C. in a sealed tubewhile consumption of starting material was monitored by TLC (mobilephase: 10% MeOH in DCM, visualization by UV). After 16 hours, thesolution was concentrated under reduced pressure and the resulting crudeproduct was triturated with EtOAc (10 mL) to afford3-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-methyl-1H-imidazol-3-iumbromide (0.15 g). MS (ESI): m/z 244.1 [M]+. 1H NMR (400 MHz, DMSO-d6) δ9.78 (s, 1H), 9.15 (s, 1H), 7.77 (s, 1H), 7.72 (s, 1H), 7.14-7.03 (m,3H), 5.28 (s, 2H), 3.91 (s, 3H), 2.17 (s, 6H).

Synthesis of1-(1-((2,6-dimethylphenyl)amino)-1-oxobutan-2-yl)pyridin-1-ium bromide

Synthesis of Intermediate 2-bromobutanoyl chloride

Thionyl chloride (150 mL) was added to 2-bromobutanoic acid (25 g, 149.7mmol, 1.0 eq) at 0° C. and the reaction mixture was subsequently stirredat 80° C. for 2 h. The reaction mixture was cooled to RT andconcentrated under reduced pressure to afford crude 2-bromobutanoylchloride (27.7 g, 99.5%) as a brown residue which was taken to next stepimmediately without further purification. ¹H NMR (400 MHz, CDCl₃) δ4.52-4.44 (m, 1H), 2.29-2.16 (m, 1H), 2.15-2.02 (m, 1H), 1.10 (t, J=7.3Hz, 3H).

Synthesis of Intermediate 2-bromo-N-(2, 6-dimethylphenyl) butanamide

A solution of 2, 6-dimethylaniline (15 g, 121.18 mmol, 1.0 eq) andpyridine (15 mL, 189.6 mmol, 1.5 eq) in DCM (400 mL) was cooled in anice bath to 0° C. To this solution was slowly added a solution of2-bromobutanoyl chloride (27.5 g, 148.6 mmol, 1.2 eq) in DCM (50 mL) andthe resulting mixture was allowed to warm to room temperature whilestirring for 2 h. The reaction mixture was adjusted to pH 5 to 6 with 2N HCl and extracted with DCM (2×200 mL). The combined organic extractswere washed with water (250 mL) and brine (200 mL), dried over Na₂SO₄,filtered and concentrated under reduced pressure. The resulting crudeproduct was triturated with n-pentane (150 mL) to afford2-bromo-N-(2,6-dimethylphenyl) butanamide (30 g). MS (ESI): m/z 272.13[M+2]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 9.69 (s, 1H), 7.15-7.02 (m, 3H),4.51 (t, J=7.3 Hz, 1H), 2.22-2.04 (m, 7H), 2.03-1.91 (m, 1H), 0.98 (t,J=7.3 Hz, 3H).

Synthesis of1-(1-((2,6-dimethylphenyl)amino)-1-oxobutan-2-yl)pyridin-1-ium bromide

To a stirred solution of 2-bromo-N-(2,6-dimethylphenyl)butanamide (0.2g, 0.7 mmol, 1.0 eq) in acetonitrile (3 mL) was added pyridine (0.12 mg,1.4 mmol, 2.0 eq) and the resulting mixture was heated in a sealed tubeat 90° C. for 36 hours. After cooling to rt, the solution was asconcentrated under reduced pressure and the resulting crude product wastriturated with EtOAc (5 mL) to afford1-(1-((2,6-dimethylphenyl)amino)-1-oxobutan-2-yl)pyridin-1-ium bromide(0.17 g) MS (ESI): m/z 269.2 [M]⁺. 1HNMR (400 MHz, DMSO-d6) δ 0.92 (t,J=7.23 Hz, 3H), 2.10 (br s, 6H), 2.28-2.45 (m, 1H), 2.59 (dt, J=13.98,6.93 Hz, 1H), 5.75 (dd, J=9.43, 5.92 Hz, 1H), 7.00-7.20 (m, 3H), 8.25(t, J=7.13 Hz, 2H), 8.73 (t, J=7.78 Hz, 1H), 9.23 (d, J=5.70 Hz, 2H),10.12 (s, 1H).

Synthesis of3-(1-((2,6-dimethylphenyl)amino)-1-oxobutan-2-yl)-1-methyl-1H-imidazol-3-iumbromide

Synthesis of 3-(1-((2,6-dimethylphenyl)amino)-1-oxobutan-2-yl)-1-methyl-1H-imidazol-3-iumbromide

Synthesis of intermediates 2-bromobutanoyl chloride and 2-bromo-N-(2,6dimethylphenyl) butanamide was as above for Compound 3. To a stirredsolution of 2-bromo-N-(2,6-dimethylphenyl) butanamide (0.2 g, 0.7 mmol,1.0 eq) in acetonitrile (3 mL) was added 1-methyl-1H-imidazole (0.12 ml,1.4 mmol, 2.0 eq) and the resulting mixture was heated in a sealed tubeat 90° C. for 36 hours. After cooling to rt, the solution was asconcentrated under reduced pressure and the resulting crude product wastriturated with EtOAc (10 mL) to afford3-(1-((2,6-dimethylphenyl)amino)-1-oxobutan-2-yl)-1-methyl-1H-imidazol-3-iumbromide (0.09 g). MS (ESI): m/z 272.2 [M]+. 1H NMR (400 MHz, DMSO-d6) δ0.95 (t, J=7.27 Hz, 3H), 2.04-2.26 (m, 7H), 2.29-2.43 (m, 1H), 3.91 (s,3H), 5.34 (dd, J=9.54, 5.72 Hz, 1H), 7.04-7.18 (m, 3H), 7.79 (t, J=1.79Hz, 1H), 7.93 (t, J=1.79 Hz, 1H), 9.40 (s, 1H), 9.97 (s, 1H).

Synthesis of1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-2-methylpyridin-1-iumbromide

Synthesis of1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-2-methylpyridin-1-iumbromide

Synthesis of intermediate 2-bromo-N-(2,6-dimethylphenyl) acetamide wasas described above for Compound 1. To a stirred solution of2-bromo-N-(2, 6-dimethylphenyl) acetamide 25 (0.2 g, 0.8 mmol, 1.0 eq)in EtOAc (5 mL) was added 2-methylpyridine (0.149 g, 1.6 mmol, 2.0 eq)and the resulting mixture was stirred at 80° C. in a sealed tube whileconsumption of starting material was monitored by TLC (mobile phase: 10%MeOH in DCM, visualization by UV). After 16 hours, the solution was asconcentrated under reduced pressure and the resulting crude product wastriturated with EtOAc (2×15 mL) to afford1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-2-methylpyridin-1-iumbromide (0.13 g) as an off white solid. MS (ESI): m/z 255.2 [M]+. 1H NMR(400 MHz, DMSO-d6) δ 2.20 (s, 6H), 2.79 (s, 3H), 5.73 (s, 2H), 6.99-7.17(m, 3H), 7.99-8.10 (m, 1H), 8.12 (d, J=7.89 Hz, 1H), 8.58 (td, J=7.78,1.10 Hz, 1H), 9.05 (d, J=5.48 Hz, 1H), 10.11 (s, 1H).

Synthesis of1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-2,6-dimethylpyridin-1-ium

Synthesis of1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-2,6-dimethylpyridin-1-iumbromide

Synthesis of intermediate 2-bromo-N-(2,6-dimethylphenyl) acetamide wasas described above for Compound 1. To a stirred solution of2-bromo-N-(2, 6-dimethylphenyl) acetamide (0.2 g, 0.8 mmol, 1.0 eq) inEtOAc (5 mL) was added 2,6-dimethylpyridine (0.17 g, 1.6 mmol, 2.0 eq)and the resulting mixture was stirred at 80° C. in a sealed tube whileconsumption of starting material was monitored by TLC (mobile phase: 10%MeOH in DCM, visualization by UV). After 32 hours, the solution wasconcentrated under reduced pressure and the resulting crude product wastriturated with EtOAc (2×10 mL) to afford1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-2,6-dimethylpyridin-1-iumbromide (0.157 g) as a white solid. ¹H NMR (400 MHz, DMSO-d6) δ 10.15(s, 1H), 8.45-8.41 (m, 1H), 7.98-7.96 (d, 2H), 7.11-7.10 (d, 3H), 5.65(s, 2H), 2.83 (s, 6H), 2.20 (s, 6H).

Synthesis of 1-(2-((4-fluoro-2, 6-dimethylphenyl)amino)-2-oxoethyl)pyridin-1-ium bromide

Synthesis of Intermediate 2-bromo-N-(4-fluoro-2, 6-dimethylphenyl)acetamide

To a stirred solution of 4-fluoro-2, 6-dimethylaniline (0.5 g, 3.6 mmol,1.0 eq) in water (10 mL) was added 2-bromoacetyl bromide (1.453 g, 7.2mmol, 2.0 eq) at 0° C. and the resulting mixture was stirred for 16 h atRT while progress of the reaction was monitored by TLC (mobile phase:50% EtOAc in petroleum ether, visualization by UV). The pH of thereaction mixture was adjusted with 15% Na₂CO₃ (aq.) solution andextracted with EtOAc (2×25 mL). The combined organic extracts were driedover Na₂SO₄, filtered and concentrated under reduced pressure to afford2-bromo-N-(4-fluoro-2, 6-dimethylphenyl) acetamide (0.45 g). MS (ESI):m/z 260.11 [M+H]+. 1H NMR (400 MHz, CDCl₃) δ 7.65 (br s, 1H), 6.81 (d,J=8.94 Hz, 2H), 4.07 (s, 2H), 2.23 (s, 6H).

Synthesis of 1-(2-((4-fluoro-2, 6-dimethylphenyl) amino)-2-oxoethyl)pyridin-1-ium bromide

To a stirred solution of 2-bromo-N-(4-fluoro-2,6-dimethylphenyl)acetamide (0.2 g, 0.8 mmol, 1.0 eq) in EtOAc (5 mL) was added pyridine(0.126 g, 1.6 mmol, 2.0 eq), and the reaction mixture was stirred for 16h at 80° C. in a sealed tube while consumption of starting material wasmonitored by TLC (mobile phase: 10% MeOH in DCM, visualization by UV).

The solution was concentrated under reduced pressure and the crudeproduct was triturated with EtOAc (15 mL) to afford 1-(2-((4-fluoro-2,6-dimethylphenyl) amino)-2-oxoethyl) pyridin-1-iumbromide (0.15 g). MS(ESI): m/z 259.2 [M]+. 1H NMR (400 MHz, DMSO-d6) δ 9.98 (s, 1H), 9.07(d, J=5.48 Hz, 2H), 8.68 (t, J=7.89 Hz, 1H), 8.22 (dd, J=7.67, 6.80 Hz,2H), 6.95 (d, J=9.43 Hz, 2H), 5.75 (s, 2H), 2.20 (s, 6H).

Synthesis of 1-(2-(mesitylamino)-2-oxoethyl)pyridin-1-ium bromide

Synthesis of Intermediate 2-bromo-N-mesitylacetamide

To a stirred solution of 2,4,6-trimethylaniline (0.5 g, 3.7 mmol, 1.0eq) in water (10 mL) was added 2-bromoacetyl bromide (1.497 g, 7.4 mmol,2.0 eq) at 0° C. and the resulting mixture was stirred for 16 h at RTwhile progress of the reaction was monitored by TLC (mobile phase: 50%EtOAc in petroleum ether, visualization by UV). The pH of the reactionmixture was adjusted with 15% Na₂CO₃ (aq.) solution and extracted withEtOAc (2×25 mL). The combined organic extracts were dried over Na₂SO₄,filtered and concentrated under reduced pressure to afford2-bromo-N-mesitylacetamide (0.65 g). MS (ESI): m/z 256.18 [M+H]. 1H NMR(400 MHz, CDCl₃) δ 7.67 (br s, 1H), 6.91 (s, 2H), 4.07 (s, 2H),2.24-2.29 (m, 3H), 2.20 (s, 6H).

Synthesis of 1-(2-(mesitylamino)-2-oxoethyl)pyridin-1-ium bromide

To a stirred solution of 2-bromo-N-mesitylacetamide (0.2 g, 0.8 mmol,1.0 eq) in ethyl acetate (5 mL) was added pyridine (0.126 g, 1.6 mmol,2.0 eq), and the reaction mixture was stirred for 16 h at 80° C. in asealed tube while the progress of the reaction was monitored by TLC(mobile phase: 10% MeOH in DCM, visualization by UV). The reactionmixture was concentrated under reduced pressure and the crude productwas triturated with EtOAc (15 mL) to afford1-(2-(mesitylamino)-2-oxoethyl)pyridin-1-ium bromide (0.13 g). MS (ESI):m/z 255.2 [M]+. 1H NMR (400 MHz, DMSO-d6) δ 9.90 (s, 1H), 9.07 (d,J=5.48 Hz, 2H), 8.68 (t, J=7.78 Hz, 1H), 8.22 (t, J=7.13 Hz, 2H), 6.89(s, 2H), 5.73 (s, 2H), 2.22 (s, 3H), 2.15 (s, 6H).

Synthesis of 1-(2-((4-chloro-2, 6-dimethylphenyl) amino)-2-oxoethyl)pyridin-1-ium bromide

Synthesis of Intermediate 2-bromo-N-(4-chloro-2, 6-dimethylphenyl)acetamide

To a stirred solution of 4-chloro-2, 6-dimethylaniline (0.5 g, 3.592mmol, 1 eq) in water (5 mL) was added 2-bromoacetyl bromide (0.797 g,3.952 mmol, 1.1 eq) at 0° C. and the resulting mixture was stirred for 1hour while progress of the reaction was monitored by TLC (mobile phase:50% EtOAc in petroleum ether, visualization by UV). The pH of thereaction mixture was adjusted with 15% Na₂CO₃ (aq.) solution andextracted with EtOAc (2×25 mL). The combined organic extracts were driedover Na₂SO₄, filtered and concentrated under reduced pressure to afford2-bromo-N-(4-chloro-2, 6-dimethylphenyl) acetamide (0.45 g). MS (ESI):m/z 276.05 [M+H]. 1H NMR (400 MHz, CDCl₃) δ 7.66 (br s, 0.5H), 7.09 (s,1H), 6.89-6.96 (m, 2H), 4.07 (s, 1H), 2.22 (s, 3H), 2.12-2.19 (m, 6H).

Synthesis of 1-(2-((4-chloro-2, 6-dimethylphenyl) amino)-2-oxoethyl)pyridin-1-ium bromide

To a stirred solution of 2-bromo-N-(4-chloro-2,6-dimethylphenyl)acetamide (0.15 g, 0.5 mmol, 1.0 eq) in EtOAc (5 mL) was added pyridine(0.085 g, 1.1 mmol, 2.0 eq), and the reaction mixture was stirred for 16h at 80° C. in a sealed tube while consumption of starting material wasmonitored by TLC (mobile phase: 10% MeOH in DCM, visualization by UV).The solution was concentrated under reduced pressure and the crudeproduct was triturated with EtOAc (15 mL) to afford1-(2-((4-chloro-2,6-dimethylphenyl)amino)-2-oxoethyl)pyridin-1-iumbromide (0.1 g) MS (ESI): m/z 275.1 [M]+. 1H NMR (400 MHz, DMSO-d6) δ10.06 (s, 1H), 9.07 (d, J=5.48 Hz, 2H), 8.68 (t, J=7.89 Hz, 1H), 8.22(t, J=7.23 Hz, 2H), 7.19 (s, 2H), 5.75 (s, 2H), 2.20 (s, 6H).

Synthesis of 1-(2-((4-methoxy-2, 6-dimethylphenyl) amino)-2-oxoethyl)pyridin-1-ium bromide

Synthesis of Intermediate 2-bromo-N-(4-methoxy-2, 6-dimethylphenyl)acetamide

To a stirred solution of 4-methoxy-2, 6-dimethylaniline (0.5 g, 3.3mmol, 1 eq) in water (5 mL) was added 2-bromoacetyl bromide (0.734 g,3.6 mmol, 1.1 eq) at 0° C. and the resulting mixture was stirred for 16hours while warming to RT and the progress of the reaction was monitoredby TLC (mobile phase: 40% EtOAc in petroleum ether, visualization byUV). The pH of the reaction mixture was adjusted with 15% Na₂CO₃ (aq.)solution and extracted with EtOAc (3×30 mL). The combined organicextracts were dried over Na₂SO₄, filtered and concentrated under reducedpressure. The resulting crude product was purified by columnchromatography (100-200 mesh silica gel; 20% EtOAc in petroleum ether)to afford 2-bromo-N-(4-methoxy-2, 6-dimethylphenyl) acetamide (0.09 g).MS (ESI): m/z 272.08 [M+H]. ¹H NMR (400 MHz, CDCl₃) δ 7.62 (br s, 1H),6.64 (s, 2H), 4.07 (s, 2H), 3.77 (s, 3H), 2.22 (s, 6H), 1.3 (m, 1H).

Synthesis of 1-(2-((4-methoxy-2, 6-dimethylphenyl) amino)-2-oxoethyl)pyridin-1-ium bromide

To a stirred solution of 2-bromo-N-(4-methoxy-2,6-dimethylphenyl)acetamide (60 mg, 0.2 mmol, 1.0 eq) in EtOAc (1 mL) was added pyridine(34 mg, 0.4 mmol, 2.0 eq), and the reaction mixture was stirred for 16 hat 80° C. in a sealed tube while consumption of starting material wasmonitored by TLC (mobile phase: 10% MeOH in DCM, visualization by UV).The solution was concentrated under reduced pressure and the crudeproduct was triturated with EtOAc (2×5 mL) to afford1-(2-((4-methoxy-2,6-dimethylphenyl)amino)-2-oxoethyl)pyridin-1-iumbromide (0.07 g) MS (ESI): m/z 271.1 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ9.82 (s, 1H), 9.07 (d, J=5.70 Hz, 2H), 8.68 (t, J=7.89 Hz, 1H), 8.22 (t,J=7.13 Hz, 2H), 6.66 (s, 2H), 5.72 (s, 2H), 3.71 (s, 3H), 2.16 (s, 6H).

Synthesis of 1-(2-((4-cyano-2, 6-dimethylphenyl) amino)-2-oxoethyl)pyridin-1-ium bromide

Synthesis of Intermediate 2-bromo-N-(4-cyano-2, 6-dimethylphenyl)acetamide

To a stirred solution of 4-amino-3, 5-dimethylbenzonitrile (0.5 g, 3.4mmol, 1.0 eq) in DCM (20 mL) was added K₂CO₃ (0.563 g, 4.08 mmol, 1.2eq) and the resulting mixture was cooled to 0° C. Next, 2-bromoacetylbromide (0.823 g, 4.08 mmol, 1.2 eq) was added drop wise and thereaction was stirred for 16 h at RT while warming to RT. Progress of thereaction was monitored by TLC (Mobile phase: 50% Ethyl acetate in petether, Rf: 0.41, Visualization by UV). The reaction mixture was basifiedwith 15% Na₂CO₃ solution at 0° C. to afford precipitate which wasfiltered and dried under vacuo to afford 2-bromo-N-(4-cyano-2,6-dimethylphenyl) acetamide (0.35 g). MS (ESI): m/z 267.36 [M+H]. 1H NMR(400 MHz, CDCl₃) δ 7.80 (br s, 1H), 7.41 (s, 2H), 4.09 (s, 2H), 2.29 (s,6H).

Synthesis of 1-(2-((4-cyano-2, 6-dimethylphenyl) amino)-2-oxoethyl)pyridin-1-ium bromide

To a stirred solution of 2-bromo-N-(4-cyano-2, 6-dimethylphenyl)acetamide (0.2 g, 0.7 mmol, 1.0 eq) in EtOAc (5 mL) was added pyridine(0.1106 g, 1.6 mmol, 2.0 eq), and the reaction mixture was stirred for16 h at 80° C. in a sealed tube while consumption of starting materialwas monitored by TLC (mobile phase: 10% MeOH in DCM, visualization byUV). The solution was concentrated under reduced pressure and the crudeproduct was triturated with EtOAc (15 mL) to afford1-(2-((4-cyano-2,6-dimethylphenyl)amino)-2-oxoethyl) pyridin-1-iumbromide (0.14 g) MS (ESI): m/z 266.1 [M]+. 1H NMR (400 MHz, DMSO-d6) δ10.32 (s, 1H), 9.08 (d, J=5.70 Hz, 2H), 8.69 (t, J=7.78 Hz, 1H), 8.23(t, J=7.23 Hz, 2H), 7.60 (s, 2H), 5.79 (s, 2H), 2.25 (s, 6H).

Synthesis of1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-phenylpyridin-1-iumbromide

Synthesis of1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-phenylpyridin-1-iumbromide

Synthesis of intermediate 2-bromo-N-(2,6-dimethylphenyl) acetamide wasas described above for Compound 1. To a stirred solution of2-bromo-N-(2,6-dimethylphenyl) acetamide (0.150 g, 0.6 mmol, 1.0 eq) inACN (2.0 ml) was added 3-phenylpyridine (0.144 g, 0.9 mmol, 1.5 eq) andthe reaction mixture was stirred at 80° C. in a sealed tube whileconsumption of starting material was monitored by TLC (mobile phase: 10%MeOH in DCM, visualization by UV). After stirring for 54 hours, thesolution was cooled to RT, concentrated under reduced pressure and thecrude product collected by filtration and triturated with EtOAc (2×5 mL)to afford1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-phenylpyridin-1-iumbromide (0.15 g). MS (ESI): m/z 317.2 [M]+. 1H NMR (400 MHz, DMSO-d6) δ10.02 (s, 1H), 9.56 (s, 1H), 9.09-8.95 (m, 2H), 8.30 (dd, J=6.1, 8.1 Hz,1H), 7.90 (d, J=7.0 Hz, 2H), 7.70-7.54 (m, 3H), 7.15-7.01 (m, 3H), 5.81(s, 2H), 2.22.

Synthesis of Compounds 13-28: Table F

Table F provides additional representative examples of the inventionwhich were synthesized as shown above or from 2-bromo-N-(2,6-dimethylphenyl) acetamide and the appropriate heterocycle according tothe described methods for the synthesis of compound 12.

TABLE F Compound Structure MS (ESI): m/z 1H NMR (400 MHz, DMSO-D6) δ 13A

269.2 [M]+ δ 10.10 (s, 1H), 9.03 (d, J = 5.48 Hz, 1H), 8.62 (td, J =7.89, 1.32 Hz, 1H), 8.12 (d, J = 8.11 Hz, 1H), 7.05-7.13 (m, 3H),8.02-8.10 (m, 1H), 5.75 (s, 2H), 3.08 (q, J = 7.38 Hz, 2H), 2.20 (s,6H), 1.36 (t, J = 7.45 Hz, 3H) 14A

317.1 [M]+ δ 9.99 (s, 1H), 9.10 (d, J = 6.80 Hz, 2H), 8.59 (d, J = 7.02Hz, 2H), 8.06- 8.17 (m, 2H), 7.61-7.73 (m, 3H), 7.03-7.15 (m, 3H), 5.72(s, 2H), 2.22 (s, 6H) 15A

291.1 [M]+ δ 10.00-10.14 (m, 2H), 8.80 (br d, J = 6.80 Hz, 1H), 8.64 (brd, J = 6.80 Hz, 1H), 8.57 (br d, J = 8.33 Hz, 1H), 8.27-8.34 (m, 1H),8.36-8.44 (m, 1H), 8.11 (br t, J = 7.56 Hz, 1H), 7.09 (s, 3H), 5.86 (s,2H), 2.23 (s, 6H) 16A

317.2 [M]+ δ 9.69 (br s, 1H), 9.21 (br d, J = 5.26 Hz, 1H), 8.75 (br t,J = 7.23 Hz, 1H), 8.29 (br s, 1H), 8.15 (br d, J = 7.45 Hz, 1H),7.48-7.81 (m, 5H), 7.04 (br s, 3H), 5.61 (br s, 2H), 1.98 (br s, 6H) 17A

269.1 [M]+ δ 10.00 (s, 1H), 9.03 (s, 1H), 8.93 (d, J = 5.92 Hz, 1H),8.58 (d, J = 8.11 Hz, 1H), 8.14 (dd, J = 7.89, 6.14 Hz, 1H), 7.02-7.15(m, 3H), 5.71 (s, 2H), 2.87 (q, J = 7.45 Hz, 2H), 2.20 (s, 6H), 1.28 (t,J = 7.56 Hz, 3H) 18A

291.1 [M]+ δ 10.21 (s, 1H), 9.61 (dd, J = 5.84, 1.31 Hz, 1H), 9.40 (d, J= 8.34 Hz, 1H), 8.54 (dd, J = 8.11, 1.19 Hz, 1H), 8.42- 8.50 (m, 1H),8.26-8.40 (m, 2H), 8.03-8.15 (m, 1H), 7.00-7.14 (m, 3H), 6.16 (s, 2H),2.20 (s, 6H) 19A

269.1 [M]+ δ 9.98 (s, 1H), 8.94 (d, J = 6.91 Hz, 2H), 8.08 (d, J = 6.68Hz, 2H), 7.02- 7.15 (m, 3H), 5.67 (s, 2H), 2.95 (q, J = 7.39 Hz, 2H),2.20 (s, 6H), 1.29 (t, J = 7.63 Hz, 3H) 20A

323.4 [M]+ δ 9.97 (s, 1 H) 9.03 (s, 1 H), 8.91 (d, J = 6.10 Hz, 1 H),8.60 (br d, J = 8.24 Hz, 1 H), 8.14 (dd, J = 7.93, 6.10 Hz, 1 H),7.03-7.19 (m, 3 H), 5.68 (s, 2 H), 2.83 (tt, J = 11.75, 3.20 Hz, 1 H),2.20 (s, 6 H), 1.79-1.94 (m, 4 H), 1.74 (br d, J = 12.82 Hz, 1 H), 1.33-1.58 (m, 4 H), 1.19-1.33 (m, 1 H) 21A

309.4 [M]+ δ 9.97 (s, 1 H), 9.02 (s, 1 H), 8.90 (d, J = 6.10 Hz, 1 H),8.60 (br d, J = 8.24 Hz, 1 H), 8.14 (dd, J = 7.93, 6.10 Hz, 1 H),7.03-7.15 (m, 3 H), 5.69 (s, 2 H), 3.21-3.29 (m, 1 H), 2.10-2.18 (m, 2H), 1.77-1.89 (m, 2 H), 1.67- 1.75 (m, 2 H), 1.54-1.65 (m, 2 H) 22A

281.4 [M]+ δ 9.97 (s, 1 H), 8.93 (s, 1 H), 8.84 (d, J = 6.10 Hz, 1 H),8.30-8.39 (m, 1 H), 8.08 (dd, J = 8.09, 5.95 Hz, 1 H), 7.03-7.15 (m, 3H), 5.65 (s, 2 H), 2.15-2.27 (m, 7 H), 1.17-1.30 (m, 2 H), 0.89-1.04 (m,2 H) 23A

345.4 [M]+ δ 10.16 (s, 1 H), 8.45 (d, J = 8.24 Hz, 1 H), 8.07 (d, J =8.24 Hz, 1 H), 7.52- 7.64 (m, 3 H), 7.44-7.51 (m, 2 H), 7.06-7.16 (m, 3H), 5.75 (s, 2 H), 2.91 (s, 3 H), 2.71 (s, 3 H), 2.21 (s, 6 H) 24A

320.4 [M]+ δ 9.82 (s, 1 H), 9.31 (d, J = 1.53 Hz, 1 H), 8.02 (d, J =1.83 Hz, 1 H), 7.54- 7.69 (m, 5 H), 7.04-7.16 (m, 3 H), 5.33 (s, 2 H),3.90 (s, 3 H), 2.20 (s, 6 H) 25A

313.4 [M]+ δ 10.01 (s, 1 H), 9.69 (s, 1 H), 9.28 (dt, J = 6.10, 1.22 Hz,1 H), 9.08 (dt, J = 8.16, 1.41 Hz, 1 H), 8.36 (dd, J = 8.09, 6.26 Hz, 1H), 7.02-7.18 (m, 3 H), 5.86 (s, 2 H), 4.47 (q, J = 7.02 Hz, 2 H), 2.21(s, 6 H), 1.38 (t, J = 7.17 Hz, 3 H) 26A

258.32 [M]+ δ 9.97 (s, 1 H), 8.39 (s, 2 H), 7.10 (d, J = 3.05 Hz, 3 H),5.64 (s, 2 H), 4.05 (s, 3 H), 2.18 (s, 6 H), 2.14 (s, 3 H). 27A

286.1 [M]+ δ 9.94 (br s, 1 H), 8.64 (br d, J = 9.78 Hz, 2 H), 6.89-7.19(m, 4 H), 5.69 (br s, 2 H), 4.32 (br d, J = 7.39 Hz, 2 H), 2.18-2.20 (m,7 H), 0.90 (br d, J = 6.32 Hz, 6 H). 28A

312.1 [M]+ δ 9.96 (s, 1 H), 8.85 (dd, J = 2.92, 0.77 Hz, 1 H), 8.61 (dd,J = 2.92, 0.77 Hz, 1 H), 6.91-7.19 (m, 4 H), 5.82 (s, 2 H), 4.33-4.54(m, 1 H), 2.19 (s, 6 H), 2.10 (br d, J = 11.92 Hz, 2 H), 1.60- 1.95 (m,5 H), 1.32-1.52 (m, 2 H), 1.10-1.32 (m, 1 H).

Synthesis of 2-(2-((2, 6-dimethylphenyl)amino)-2-oxoethyl)-1-phenyl-1H-pyrazol-2-ium formate

To a stirred solution of 2-bromo-N-(2, 6-dimethylphenyl) acetamide (500mg, 2.06 mmol) in ACN (10 mL) was added 1-phenyl-1H-pyrazole (595 mg,4.12 mmol) in a microwave vial. The resulting reaction mixture wasstirred at 100° C. in a microwave reactor (CEM Corporation, Matthews,N.C.) for 2 h. The reaction mixture was concentrated under reducedpressure to afford crude product which was purified by flash columnchromatography (silica gel 100-200 mesh; elution with 10%-25% MeOH/DCMgradient). The collected pure fractions were concentrated under reducedpressure to afford (120 mg, LCMS 48%) which was purified by flash columnchromatography (silica gel 100-200 mesh; elution with 10%-25% MeOH/DCMgradient) and then further purified by reverse phase Prep-HPLC method(column: X-Select CSH C18 (250*19) mm, 5 u, temperature: ambient, mobilephase (A): 0.1% formic acid in water; mobile phase (B): ACN; (T/% B)0/10, 2/10, 10/35, 14/35, 14.2/98, 17/98, 17.2/10, 20/10, flow: 13mL/min). Pure fractions were combined and concentrated by lyophilizationto deliver 2-(2-((2, 6-dimethylphenyl)amino)-2-oxoethyl)-1-phenyl-1H-pyrazol-2-ium (20.3 mg). MS (ESI): m/z306.2 [M]+. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.06 (s, 1H), 8.92 (dd,J=9.54, 2.69 Hz, 2H), 8.51 (s, 1H), 7.64-7.85 (m, 5H), 7.21 (t, J=2.69Hz, 1H), 6.93-7.10 (m, 3H), 5.58 (s, 2H), 1.87 (s, 6H).

Synthesis of 2-(2-((2, 6-dimethylphenyl)amino)-2-oxoethyl)-1-(2-(ethylamino)-2-oxoethyl)-1H-pyrazol-2-iumbromide

To a solution of 2-bromo-N-(2, 6-dimethylphenyl) acetamide (100 mg,0.413 mmol) in ACN (2 mL) was added N-ethyl-2-(1H-pyrazol-1-yl)acetamide (94.9 mg, 0.619 mmol) at RT and the resulting reaction mixturewas stirred at 90° C. for 72 h in a sealed tube as progress of thereaction was monitored by TLC (10% MeOH in DCM, visualization by UV).The reaction mixture was cooled to RT and concentrated under reducedpressure. Crude product was triturated with EtOAc (2×8 mL) to affordproduct 2-(2-((2, 6-dimethylphenyl)amino)-2-oxoethyl)-1-(2-(ethylamino)-2-oxoethyl)-1H-pyrazol-2-iumbromide (90 mg). Mass (ESI): m/z 315.1 [M]⁺. UPLC: 97.79%. ¹H NMR (400MHz, DMSO-d₆) δ ppm 9.92 (s, 1H), 8.68 (dd, J=2.86, 0.95 Hz, 1H),8.53-8.64 (m, 2H), 7.05-7.16 (m, 3H), 7.01 (t, J=2.98 Hz, 1H), 5.58 (s,2H), 5.28 (s, 2H), 3.09-3.25 (m, 2H), 2.18 (s, 6H), 1.09 (t, J=7.27 Hz,3H).

Synthesis of Compounds 31-105: Table G

The following Table G provides additional representative examples of theinvention which were synthesized as shown above from either2-bromo-N-(2, 6-dimethylphenyl) acetamide or 2-chloro-N-(2,6-dimethylphenyl) acetamide and the appropriate heterocycle according tothe described methods. Compounds were purified by trituration or reversephase prep HPLC.

TABLE G Compound Structure MS (ESI): m/z  31A

283.27 [M]⁺  32A

297.37 [M]⁺  33A

312.50 [M]⁺  34A

331.41 [M]⁺  35A

280.33 [M]⁺  36A

286.38 [M]⁺  37A

306.33 [M]⁺  38A

320.39 [M]⁺  39A

255.29 [M]⁺  40A

298.34 [M]⁺  41A

312.43 [M]⁺  42A

257.31 [M]⁺  43A

271.32 [M]⁺  44A

259.29 [M]⁺  45A

275.28 [M]⁺  46A

319.33 [M]⁺  47A

348.17 [M]⁺  48A

298.37 [M]⁺  49A

256.3 [M]⁺  50A

284.38 [M]⁺  51A

310.42 [M]⁺  52A

266.31 [M]⁺  53A

309.37 [M]⁺  54A

294.34 [M]⁺  55A

270.35 [M]⁺  56A

242.28 [M]⁺  57A

272.37 [M]⁺  58A

272.37 [M]⁺  59A

302.5 [M]⁺  60A

316.45 [M]⁺  61A

316.38 [M]⁺  62A

315.44 [M]⁺  63A

314.41 [M]⁺  64A

340.33 [M]⁺  65A

277.21 [M]⁺  66A

309.21 [M]⁺  67A

309.18 [M]⁺  68A

270.1.0 [M]⁺  69A

269.0 [M]⁺  70A

283 [M]⁺  71A

298.1 [M]⁺  72A

284 [M]⁺  73A

284.1 [M]⁺  74A

300.1 [M]⁺  75A

344.0 [M]⁺  76A

330.32 [M + H]  77A

335.0 [M]⁺  78A

289.0 [M]⁺  79A

335.0 [M]⁺  80A

330.06, [M]⁺  81A

320.1 [M]⁺  82A

286.1 [M]⁺  83A

286.1[M]⁺  84A

330.35 [M]⁺  85A

330.1, [M]⁺  86A

330.0 [M]⁺  87A

299.0 [M]⁺  88A

347 [M]⁺  89A

334.9 [M]⁺  90A

352.0 [M]⁺  91A

324.0 [M]⁺  92A

320.1 [M]⁺  93A

258.1 [M]⁺  94A

272.1 [M]⁺  95A

272.1 [M]⁺  96A

320 [M]⁺  97A

296.0 [M]⁺  98A

301.1 [M]⁺  99A

334.27 [M]⁺ 100A

367.0 [M]⁺ 101A

286.33 [M]⁺ 102A

286.1 [M]⁺ 103A

380.9 [M]+ 104A

272 [M]⁺ 105A

257.31 [M]⁺

Synthesis of1-(1-((2,6-dimethylphenyl)amino)-1-oxo-3-phenylpropan-2-yl)pyridin-1-iumchloride

Synthesis of 2-chloro-N-(2, 6-dimethylphenyl)-3-phenylpropanamide

To a stirred solution of 2-bromo-3-phenylpropanoic acid (0.2 g, 0.873mmol), EDC.HCl (0.836 g, 4.365 mmol) and DMAP (0.085 g, 0.698 mmol) inDCM (5 ml) was added 2, 6-dimethylaniline (0.116 g, 0.960 mmol) at roomtemperature and the resulting reaction mass was stirred at roomtemperature for 60 h in a sealed tube as progress of the reaction wasmonitored by TLC (20% EtOAc-Hexane, Visualization: UV). The reactionmixture was concentrated under reduced pressure to afford crude productwhich was diluted with EtOAc (100 ml), washed with water (20 ml×3),dried over anhydrous Na₂SO₄ and concentrated under reduced pressure toafford crude product 0.26 g. The crude product was purified by normalphase flash chromatography (eluted with 10%-50% of EtOAc/Pet ether) toafford 2-chloro-N-(2, 6-dimethylphenyl)-3-phenylpropanamide (0.1 g) asan off-white solid. MS (ESI): m/z 287.82 [M+H]⁺. ¹H NMR (400 MHz,DMSO-d₆) δ ppm 9.61 (s, 1H), 7.35-7.23 (m, 5H), 7.08-6.99 (m, 3H), 4.83(t, 1H), 3.39-3.35 (m, 1H), 3.21-3.14 (m, 1H), 1.92 (s, 6H).

Synthesis of 1-1-(1-((2, 6-dimethylphenyl)amino)-1-oxo-3-phenylpropan-2-yl) pyridin-1-ium chloride

To a stirred solution of 2-chloro-N-(2,6-dimethylphenyl)-3-phenylpropanamide (0.05 g, 0.174 mmol) in EtOAc (5ml) was added pyridine (0.059 g, 0.745 mmol) at room temperature and theresulting reaction was stirred at 120° C. for 16 hours in a sealed tubeas progress of the reaction was monitored by TLC (10% MeOH in DCM,Visualization: UV). The reaction mixture was concentrated under reducedpressure to afford crude product which was triturated with Ethyl acetate(20 ml×3) to afford 1-1-(1-((2, 6-dimethylphenyl)amino)-1-oxo-3-phenylpropan-2-yl) pyridin-1-ium chloride (42.3 mg) as anoff white solid. MS (ESI): m/z 331.25 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δppm 10.50 (s, 1H), 9.28 (d, 2H), 8.64-8.60 (m, 1H), 8.18-8.14 (m, 2H),7.34-7.19 (m, 5H), 7.13-7.05 (m, 3H), 6.33-6.29 (m, 1H), 4.03-3.98 (m,1H), 3.73-3.67 (m, 1H), 2.03 (s, 6H).

Synthesis of Compounds 107-108: Table H

The following Table H provides additional representative examples of theinvention which were synthesized from the appropriately substituted2-bromo-propanoic acid, 2,6-dimethylaniline and pyridine.

TABLE H Compound Structure MS (ESI): m/z 107A

283.2 [M]⁺ 108A

297.1 [M]⁺

Synthesis of 1, 2-bis (2-((2, 6-dimethylphenyl)amino)-2-oxoethyl)-1H-pyrazol-2-ium bromide

A solution of 2-bromo-N-(2, 6-dimethylphenyl) acetamide (200 mg, 0.8260mmol) and 1-(tert-butyl)-1H-pyrazole (0.5 mL) in sealed tube was stirredat 120° C. for 16 h as progress of the reaction was monitored by TLC(10% MeOH in DCM, Detection: UV). The reaction mixture was cooled roomtemperature and concentrated under reduced pressure to afford crudeproduct which was purified by column chromatography (eluted with 20%MeOH in DCM) to afford 1, 2-bis(2-((2, 6-dimethylphenyl)amino)-2-oxoethyl)-1H-pyrazol-2-ium bromide (111.9 mg). MS (ESI): m/z391.0 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.17 (s, 2H), 8.74 (d, 2H),7.04-7.12 (m, 7H), 5.65 (s, 4H), 2.19 (s, 12H).

Synthesis of1,3-bis(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1H-imidazol-3-iumbromide

To a stirred solution of 2-bromo-N-(2,6-dimethylphenyl)acetamide (0.15g, 0.619 mmol) in Acetonitrile (2.0 ml) was added 1H-imidazole (0.084 g,1.238 mmol) at room temperature and the resulting reaction mixture wasstirred for 16 h at 90° C. as progress of the reaction mixture wasmonitored by TLC (10% MeOH in DCM, visualization: UV). The reaction wasconcentrated under reduced pressure to afford crude product which wastriturated with 5% MeOH in DCM (50 ml) to afford1,3-bis(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1H-imidazol-3-iumbromide (56 mg) as an off white solid. Mass (ESI): m/z 391.38 [M]⁺. ¹HNMR (400 MHz, DMSO-d₆) δ ppm 9.82 (s, 2H), 9.24 (s, 1H), 7.80 (d, 2H),7.12-7.06 (s, 6H), 5.34 (s, 4H), 2.17 (s, 12H).

Synthesis of 1-cyclohexyl-2-(2-(2,6-dimethylphenoxy)-2-oxoethyl)-1H-pyrazol-2-ium bromide

Synthesis of Intermediate 2, 6-dimethylphenyl 2-bromoacetate

To a stirred solution of 2, 6-dimethyl phenol (0.5 g, 4.092 mmol) inacetonitrile (5 ml) was added pyridine (0.647 g, 8.184 mmol) and2-bromoacetyl bromide (1.23 g, 6.138 mmol) at 0° C. and the resultingreaction mixture was stirred at 0° C. for 15 min as progress of thereaction was monitored by TLC (10% EtOAc in Pet ether, Visualization:UV). The reaction mixture was diluted with water (20 ml), extracted withethyl acetate (2×25 ml), washed with brine (30 ml), dried over Na₂SO₄and concentrated under reduced pressure to afford 2, 6-dimethylphenyl2-bromoacetate (500 mg) as a pale yellow liquid. ¹H NMR (400 MHz, CDCl₃)δ ppm 7.07 (s, 3H), 4.07 (s, 2H), 2.18 (s, 6H).

Synthesis of 1-cyclohexyl-2-(2-(2,6-dimethylphenoxy)-2-oxoethyl)-1H-pyrazol-2-ium bromide

To a stirred solution of 2, 6-dimethylphenyl 2-bromoacetate (0.5 g,2.056 mmol) in ACN (5 ml) was added 1-cyclohexyl-1H-pyrazole (0.617 g,4.112 mmol) at room temperature and the resulting reaction mixture washeated to 90° C. for 16 h as progress of the reaction was monitored byTLC (10% MeOH in DCM, Visualization: UV). The reaction mixture wascooled to room temperature and concentrated under reduced pressure toafford crude product which was purified by normal phase flashchromatography (10%-50% of MeOH in DCM) to afford1-cyclohexyl-2-(2-(2,6-dimethylphenoxy)-2-oxoethyl)-1H-pyrazol-2-iumbromide (20 mg) as an off white solid Mass (ESI): m/z 313.2 [M]+. ¹H NMR(400 MHz, DMSO-d6) δ ppm 8.95 (d, 1H), 8.68 (d, 1H), 7.17-7.11 (m, 4H),6.32 (s, 2H), 4.66-4.61 (m, 1H), 2.16 (s, 6H), 2.05-2.02 (m, 2H),1.87-1.78 (m, 4H), 1.71-1.67 (m, 1H), 1.44-1.41 (m, 2H), 1.25-1.22 (m,1H).

Synthesis of Compounds 112-130: Table I

The following Table I provides additional representative examples of theinvention which were synthesized as shown above from 2-bromoacetylbromide and the appropriately substituted aniline and heterocycleaccording to the described methods. Compounds were purified bytrituration or reverse phase prep HPLC.

TABLE I Compound Structure MS (ESI): m/z 112A

329.2 [M]⁺ 113A

339.2 [M]⁺ 114A

334.3 [M]⁺ 115A

331.2 [M]⁺ 116A

335.2 [M]⁺ 117A

351.3[M]⁺ 118A

342.1 [M]⁺ 119A

326.2 [M]⁺ 120A

346.2 [M]⁺ 121A

337.3 [M]⁺ 122A

331.2 [M]⁺ 123A

321.2 [M]⁺ 124A

337.2 [M]⁺ 125A

328.1 [M]⁺ 126A

333.2 [M]⁺ 127A

316.2 [M]⁺ 128A

323.2 [M]⁺ 129A

328.2 [M]⁺

Example 2—Representative Compounds

Representative compounds according to the invention and theirenantiomers and pharmaceutically acceptable salts thereof are describedbelow, wherein Y⁻ is a pharmaceutically acceptable anion as definedabove and Z is either a heteroaryl structure selected from one of thestructures in the tables that follow. The compounds can be madeaccording to the methods generally described above.

No. Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

67

58

TABLE 1 Representative Z Structures No. Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

TABLE 2 Representative Z Structures No. Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

63

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

TABLE 3 Representative Z Structures No. Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46.

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

TABLE 4 Representative Z Structures No. Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

TABLE 5 Representative Z Structures No. Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

45

45

TABLE 6 Representative Z Structures No. Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46.

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

Preferred compounds according to the invention and their enantiomers andpharmaceutically acceptable salts thereof are represented by Formula(IV),

wherein the preferred compounds consist of substituent combinationsR^(C), R^(D), and Z as defined in Table 7, and Y⁻ is a pharmaceuticallyacceptable anion as defined above. The compounds can be made accordingto the methods generally described above.

TABLE 7 Preferred Combinations of R^(C), R^(D), and Z Subtituentsaccording to Formula (IV). Combination Number R^(C) R^(D) Z 1 H H

2 Cl H

3 CH₃ H

4 H CH₂CH₃

5 Cl CH₂CH₃

6 CH₃ CH₂CH₃

7 H (CH₂)₂CH₃

8 Cl (CH₂)₂CH₃

9 CH₃ (CH₂)₂CH₃

10 H H

11 Cl H

12 CH₃ H

13 H CH₂CH₃

14 Cl CH₂CH₃

15 CH₃ CH₂CH₃

16 H (CH₂)₂CH₃

17 Cl (CH₂)₂CH₃

18 CH₃ (CH₂)₂CH₃

19 H H

20 Cl H

21 CH₃ H

22 H CH₂CH₃

23 Cl CH₂CH₃

24 CH₃ CH₂CH₃

25 H (CH₂)₂CH₃

26 Cl (CH₂)₂CH₃

27 CH₃ (CH₂)₂CH₃

28 H H

29 Cl H

30 CH₃ H

31 H CH₂CH₃

32 Cl CH₂CH₃

33 CH₃ CH₂CH₃

34 H (CH₂)₂CH₃

35 Cl (CH₂)₂CH₃

36 CH₃ (CH₂)₂CH₃

37 H H

38 Cl H

39 CH₃ H

40 H CH₂CH₃

41 Cl CH₂CH₃

42 CH₃ CH₂CH₃

43 H (CH₂)₂CH₃

44 Cl (CH₂)₂CH₃

45 CH₃ (CH₂)₂CH₃

46 H H

47 Cl H

48 CH₃ H

49 H CH₂CH₃

50 Cl CH₂CH₃

51 CH₃ CH₂CH₃

52 H (CH₂)₂CH₃

53 Cl (CH₂)₂CH₃

54 CH₃ (CH₂)₂CH₃

55 H H

56 Cl H

57 CH₃ H

58 H CH₂CH₃

59 Cl CH₂CH₃

60 CH₃ CH₂CH₃

61 H (CH₂)₂CH₃

62 Cl (CH₂)₂CH₃

63 CH₃ (CH₂)₂CH₃

64 H H

65 Cl H

66 CH₃ H

67 H CH₂CH₃

68 Cl CH₂CH₃

69 CH₃ CH₂CH₃

70 H (CH₂)₂CH₃

71 Cl (CH₂)₂CH₃

72 CH₃ (CH₂)₂CH₃

73 H H

74 Cl H

75 CH₃ H

76 H CH₂CH₃

77 Cl CH₂CH₃

78 CH₃ CH₂CH₃

79 H (CH₂)₂CH₃

80 Cl (CH₂)₂CH₃

81 CH₃ (CH₂)₂CH₃

82 H H

83 Cl H

84 CH₃ H

85 H CH₂CH₃

86 Cl CH₂CH₃

87 CH₃ CH₂CH₃

88 H (CH₂)₂CH₃

89 Cl (CH₂)₂CH₃

90 CH₃ (CH₂)₂CH₃

91 H H

92 Cl H

93 CH₃ H

94 H CH₂CH₃

95 Cl CH₂CH₃

96 CH₃ CH₂CH₃

97 H (CH₂)₂CH₃

98 Cl (CH₂)₂CH₃

99 CH₃ (CH₂)₂CH₃

100 H H

101 Cl H

102 CH₃ H

103 H CH₂CH₃

104 Cl CH₂CH₃

105 CH₃ CH₂CH₃

106 H (CH₂)₂CH₃

107 Cl (CH₂)₂CH₃

108 CH₃ (CH₂)₂CH₃

109 H H

110 Cl H

111 CH₃ H

112 H CH₂CH₃

113 Cl CH₂CH₃

114 CH₃ CH₂CH₃

115 H (CH₂)₂CH₃

116 Cl (CH₂)₂CH₃

117 CH₃ (CH₂)₂CH₃

118 H H

119 Cl H

120 CH₃ H

121 H CH₂CH₃

122 Cl CH₂CH₃

123 CH₃ CH₂CH₃

124 H (CH₂)₂CH₃

125 Cl (CH₂)₂CH₃

126 CH₃ (CH₂)₂CH₃

127 H H

128 Cl H

129 CH₃ H

130 H CH₂CH₃

131 Cl CH₂CH₃

132 CH₃ CH₂CH₃

133 H (CH₂)₂CH₃

134 Cl (CH₂)₂CH₃

135 CH₃ (CH₂)₂CH₃

136 H H

137 Cl H

138 CH₃ H

139 H CH₂CH₃

140 Cl CH₂CH₃

141 CH₃ CH₂CH₃

142 H (CH₂)₂CH₃

143 Cl (CH₂)₂CH₃

144 CH₃ (CH₂)₂CH₃

145 H H

146 Cl H

147 CH₃ H

148 H CH₂CH₃

149 Cl CH₂CH₃

150 CH₃ CH₂CH₃

151 H (CH₂)₂CH₃

152 Cl (CH₂)₂CH₃

153 CH₃ (CH₂)₂CH₃

154 H H

155 Cl H

156 CH₃ H

157 H CH₂CH₃

158 Cl CH₂CH₃

159 CH₃ CH₂CH₃

160 H (CH₂)₂CH₃

161 Cl (CH₂)₂CH₃

162 CH₃ (CH₂)₂CH₃

163 H H

164 Cl H

165 CH₃ H

166 H CH₂CH₃

167 Cl CH₂CH₃

168 CH₃ CH₂CH₃

169 H (CH₂)₂CH₃

170 Cl (CH₂)₂CH₃

171 CH₃ (CH₂)₂CH₃

172 H H

173 Cl H

174 CH₃ H

175 H CH₂CH₃

176 Cl CH₂CH₃

177 CH₃ CH₂CH₃

178 H (CH₂)₂CH₃

179 Cl (CH₂)₂CH₃

180 CH₃ (CH₂)₂CH₃

181 H H

182 Cl H

183 CH₃ H

184 H CH₂CH₃

185 Cl CH₂CH₃

186 CH₃ CH₂CH₃

187 H (CH₂)₂CH₃

188 Cl (CH₂)₂CH₃

189 CH₃ (CH₂)₂CH₃

190 H H

191 Cl H

192 CH₃ H

193 H CH₂CH₃

194 Cl CH₂CH₃

195 CH₃ CH₂CH₃

196 H (CH₂)₂CH₃

197 Cl (CH₂)₂CH₃

198 CH₃ (CH₂)₂CH₃

199 H H

200 Cl H

201 CH₃ H

202 H CH₂CH₃

203 Cl CH₂CH₃

204 CH₃ CH₂CH₃

205 H (CH₂)₂CH₃

206 Cl (CH₂)₂CH₃

207 CH₃ (CH₂)₂CH₃

208 H H

209 Cl H

210 CH₃ H

211 H CH₂CH₃

212 Cl CH₂CH₃

213 CH₃ CH₂CH₃

214 H (CH₂)₂CH₃

215 Cl (CH₂)₂CH₃

216 CH₃ (CH₂)₂CH₃

217 H H

218 Cl H

219 CH₃ H

220 H CH₂CH₃

221 Cl CH₂CH₃

222 CH₃ CH₂CH₃

223 H (CH₂)₂CH₃

224 Cl (CH₂)₂CH₃

225 CH₃ (CH₂)₂CH₃

226 H H

227 Cl H

228 CH₃ H

229 H CH₂CH₃

230 Cl CH₂CH₃

231 CH₃ CH₂CH₃

232 H (CH₂)₂CH₃

233 Cl (CH₂)₂CH₃

234 CH₃ (CH₂)₂CH₃

235 H H

236 Cl H

237 CH₃ H

238 H CH₂CH₃

239 Cl CH₂CH₃

240 CH₃ CH₂CH₃

241 H (CH₂)₂CH₃

242 Cl (CH₂)₂CH₃

243 CH₃ (CH₂)₂CH₃

244 H H

245 Cl H

246 CH₃ H

247 H CH₂CH₃

248 Cl CH₂CH₃

249 CH₃ CH₂CH₃

250 H (CH₂)₂CH₃

251 Cl (CH₂)₂CH₃

252 CH₃ (CH₂)₂CH₃

253 H H

254 H CH₂CH₃

255 H (CH₂)₂CH₃

256 H H

257 H CH₂CH₃

258 H (CH₂)₂CH₃

259 H H

260 H CH₂CH₃

261 H (CH₂)₂CH₃

262 H H

263 H CH₂CH₃

264 H (CH₂)₂CH₃

Example 3—Inhibition of Nav1.7 Current

Representative compounds of the invention were synthesized according tothe described methods and tested for the ability to inhibitvoltage-gated sodium channels.

Cell Culture

NaV1.7 was expressed upon induction with tetracycline. Cells werecultured in DVEM containing 10% dialyzed Fetal Bovine Serum (VWR,Radnor, Pa.), 1% Glutamax (VWR, Radnor, Pa.), 1% Penicillin-Streptomycin(VWR, Radnor, Pa.), 100 mg/L Hygromycin (Thermo Fisher Scientific,Waltham, Mass. and 5 mg/L Blasticidin (Alfa Aesar, Haverhill, Mass.).Cells were grown and maintained at 37° C. in a humidified environmentcontaining 10% CO₂ in air. Cells were detached from the culture flaskfor passage and harvested using 0.05% Trypsin-EDTA (Thermo FisherScientific, Waltham, Mass.). To induce NaV1.7, cells were induced withtetracycline (0.1-1 μg/mL, IBI Scientific, Peosta, Iowa) the day beforerecording and plated onto 24-well plates. Cells were washed with DPBS(VWR, Radnor, Pa.), trypsinized and then triturated five times in 10 mLof growth media to break apart cell aggregates. For one 24-well plate, 2mL of cell suspension was mixed with 23 mL of fresh growth media and0.1-1 μg/mL tetracycline added. 1 ml of mixed media with cells was thenadded to each well of a 24-well plate, with a 12 mm coverslip alreadyplaced in the bottom of the well. Cells were then incubated in 37° C.and 10% CO₂ overnight.

Patch Clamp Solutions & Drugs

The intracellular solution contained the following (in mM) CsCl 135,NaCl 10, EGTA 10, HEPES 10, MgCl₂ 2, adjusted to pH 7.2 with CsOH. Theexternal solution was a normal Ringer solution containing (in mM) NaCl155, HEPES 10, glucose 10, KCl 3.5, CaCl₂) 1.5, MgCl₂ 1 adjusted to pH7.4 with NaOH. CsCl is from Alfa Aesar, Haverhill, Mass. All otherchemicals are from Sigma-Aldrich, St. Louis, Mo. In order to test thedegree of internal block by test compounds the compounds were dissolvedin internal solution at the indicated test concentration. In controlexperiments the internal solution did not contain any compound. In orderto test the degree of external block by test compounds the compoundswere dissolved in external solution at the indicated test concentration.

Whole Cell Patch Clamp Protocol

18-24 hours after cells were induced with tetracycline, coverslips wereplaced into a chamber filled with Normal Ringer solution at roomtemperature and the chamber placed on a microscope. Pipettes were pulledfrom borosilicate glass on a P97 puller (Sutter Instrument, Novato,Calif.) and polished with a MF-830 Microforge (Narishige InternationalUSA, Inc, Amityville, N.Y.) to have a resistance of 1.5-2.5 MΩ whenfilled with CsCl internal solution at room temperature. Healthy cells(those that are round and translucent with no visible blemishes) werechosen for seal formation. A seal was formed between the pipette and thecell, and a brief pulse of suction was used to “break in” and establishthe whole-cell configuration. The membrane potential was held at −100 mVbefore the voltage protocol began. Only cells with series resistancebetween 1.5-5 MΩ were retained for analysis. The voltage protocol was asfollows: Cells were held at −100 mV for 12 ms followed by ahyperpolarizing step to −105 mV for 12 ms to monitor the leak. Cellswere then stepped back to −100 mV for 40 ms. Cells were then depolarizedto −20 mV for 10 ms and then returned to −100 mV for 26 ms.

Internal Block by Test Compounds

Once the recording was started, the voltage protocol was run at 30second intervals for 5 minutes to get a stable baseline. This wasfollowed by four 30-second periods of 5 Hz stimulation of the samevoltage protocol separated by 1 minute of rest which was then followedby 0.33 Hz stimulation after the last train. Currents were recordedusing PatchMaster software with Heka EPC10 (HEKA Electronics, Lambrecht,Germany). Only cells with inward current amplitudes at −20 mV between400 pA and 4 nA were accepted. In addition, cells having leak currentsgreater than 10% of their current amplitudes were discarded.

Data Analysis: Internal Block

The data was plotted using the Patchmaster software (HEKA Electronics,Lambrecht, Germany) and analyzed by plotting the minimum current duringthe voltage step to −20 mV (peak inward current) as a function of time.In order to determine the degree of rundown over the course of anexperiment, the average peak inward current amplitude (2-3 points)before 5 Hz stimulation was designated as the baseline (I_(baseline)).The average peak inward current during the last 2 second of the last 5Hz train was measured (I_(test)). The control fraction current remainingwas calculated by dividing I_(test) by I_(baseline). On each recordingday three cells were tested with control internal solution and theaverage fraction of current remaining calculated (Ctrl fractioncurrent).

To determine the % block produced by test compounds applied internallythe following was done. The average peak inward current amplitude (2-3points) before 5 Hz stimulation was designated as 0% block(I_(0% block)). To correct for the current change under controlconditions, I_(0% block) was multiplied by the average Ctrl fractioncurrent remaining to get the corrected 0% block current. The averagepeak inward current during the last 2 seconds of the last 5 Hz train wasdesignated as the unblocked current (I_(unblocked)). The % block wascalculated using the following equation:(1−I_(unblocked)/(I_(0% block)*Ctrl fraction current remaining)×100).

Representative examples of the invention were tested for intracellularinhibition of NaV 1.7. Activity Range is % inhibition at 32 μM: “++++”(>95%), “+++” 95-70%, “++” (70-40%) or “+” (<40%). The results arepresented below.

TABLE 1 Nav1.7 Nav1.7 Nav1.7 Com- Intracellular Com- Intracellular Com-Intracellular pound Inhibition pound Inhibition pound Inhibition1A + + + 9A + 17A + + + 2A + + 10A + 18A + + + + 3A + + + 11A +19A + + + 4A + + 12A + + + + 20A + + + 5A + + + 13A + + + 21A + + + +6A + + + + 14A + 22A 7A + 15A + + + + 23A + + + + 8A + 16A + + + +24A + + +

Representative examples of the invention were tested for intracellularinhibition of NaV 1.7. Activity Range is 0% inhibition at 10 μM: “++++”(>95%), “+++” 95-70%, “++” (70-40%) or “+” (K 40%). The results arepresented below.

TABLE K Nav1.7 Nav1.7 Nav1.7 Com- Intracellular Com- Intracellular Com-Intracellular pound Inhibition pound Inhibition pound Inhibition12A + + + + 46 + + + 73 + + + + 16A + + + + 47 + + + 75 + + + +17A + + + 48 + + + + 76 + + + 25A + + + 49 + + + 77 + + + + 26A + + +50 + + + + 79 + + + + 27A + + + 51 + + + + 81 + + + 28A + + + + 52 + + +83 + + + 29A + + 53 + 84 + + + 30A + + + 54 + + + 89 + + 31A + + +55 + + + 94 + + + 32 + + + 56 + 95 + 33 + + + 57 + + + 96 + + + 34 + + +58 + + + 97 + + + 35 + + 59 + + + 98 + + + 36 + + + 60 + + 101 + + +37 + + + 61 + + 103 + + + + 38 + + + 62 + 104 + + + 39 + + + 63 + +106 + + + + 40 + + + 64 + 108 + + + 41 + + 64 + + + 109 + + + + 42 +66 + + + 110 + + + 43 + + + 67 + + 111 + + + + 44 + + + 69 + + 101 + + +45 + + + 71 + + + + 103 + + + +

External Block by Test Compounds

Once the recording was started, the voltage protocol was run at 30second intervals for 5 minutes to get a stable baseline. This isfollowed by 5 Hz stimulation of the same voltage protocol run until theend of experiment. The test compound is added during the 5 Hzstimulation train making sure to wait until the cell shows stablecurrent rundown rate before addition of the compound. The test compoundis added for 5 minutes before washing out with normal Ringer's solution.Currents were recorded using PatchMaster software with Heka EPC10 (HEKAElectronics, Lambrecht, Germany). Only cells with inward currentamplitudes at −20 mV between 400 pA and 4 nA were accepted. In addition,cells having leak currents greater than 10% of their current amplitudeswere discarded.

Data Analysis: External Block

The data was plotted using the Patchmaster software (HEKA Electronics,Lambrecht, Germany) and analyzed by plotting the minimum current duringthe voltage step to −20 mV (peak inward current) as a function of time.To determine the % block produced by test compounds applied externallythe following was done. After the stable current rundown rate wasestablished during the 5 Hz stimulation train, the Rate_(rundown) wascalculated by dividing the change in peak current amplitude by time. Theaverage peak inward current amplitude (2-3 seconds) before addition ofcompound was used to determine 0% block (I_(0% block)). To correct forthe rundown, I_(0% block) is subtracted by the (Rate_(rundown)*5 min) toget the corrected 0% block current. The average peak inward currentduring the last 2-3 seconds of the 5 minutes of compound applicationtime before washing is the unblocked current (I_(unblocked)). The %block was then calculated using the following equation: Fraction currentblock=1−I_(unblocked)/(I_(0% block)−Rate_(rundown)*5 min).

Representative examples of the invention were tested for extracellularinhibition of NaV 1.7. Activity Range is % inhibition: “++++” (>95%),“+++” 95-70%, “++” (70-40%) or “+” (<40%). The results are presentedbelow.

TABLE L Compound Test Concentration Nav1.7 Extracellular Inhibition 6A1,000 μM + + 12A 30 μM + 13A 30 μM + 16A 100 μM + 17A 30 μM + 25A 30μM + + 26A 30 μM + 27A 30 μM + 28A 30 μM + 29A 30 μM + 30A 30 μM + 751,000 μM + 76 1,000 μM + 109 1,000 μM +

Automated Patch Clamp: Cell Culture

NaV1.7 was expressed in HEK293 cells upon induction with tetracycline.Cells were cultured in DMEM containing 10% dialyzed Fetal Bovine Serum(VWR, Radnor, Pa.), 1% Glutamax (VWR, Radnor, Pa.), 1%Penicillin-Streptomycin (VWR, Radnor, Pa.), 100 mg/L Hygromycin (ThermoFisher Scientific, Waltham, Mass. and 5 mg/L Blasticidin (Alfa Aesar,Haverhill, Mass.). Cells were grown and maintained at 37° C. in ahumidified environment containing 10% CO₂ in air. Cells were detachedfrom the culture flask for passage and harvested using 0.05%Trypsin-EDTA (Thermo Fisher Scientific, Waltham, Mass.). To induceNaV1.7, cells were induced with tetracycline (0.1-1 μg/mL, IBIScientific, Peosta, Iowa) the day before recording.

Before experiments, cells were washed with DPBS (VWR, Radnor, Pa.),digested with Detachin (VWR Radnor, Pa.) and then triturated 10 times inCHO Serum-Free Media (VWR Radnor, Pa.) to resuspend the cells and tobreak apart cell aggregates. Cells were counted and the finalconcentration was set at 2-5 million cells per mL.

Patch Clamp Solutions & Drugs

The intracellular solution contained the following: 140 mM CsF, 1 mM/5mM EGTA/CsOH, 10 mM HEPES, 10 mM NaCl, adjusted to pH 7.3 with CsOH, andosmolality to 320 with sucrose. The external solution contained thefollowing: 145 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl₂), 10 mM HEPES,10 mM Glucose, adjusted to pH 7.4 with CsOH and osmolality to 305 withsucrose. All chemicals are from Sigma-Aldrich, St. Louis, Mo. In orderto test the degree of internal block by test compounds, the compoundswere dissolved in internal solution at the indicated test concentration.In control experiments the internal solution did not contain anycompound. In order to test the degree of external block by testcompounds the compounds were dissolved in external solution at theindicated test concentration.

Automated Patch Clamp Protocol

Automated Patch Clamps were performed on Qube 384 (Sophion Bioscience,Woburn Mass.) with multihole Qchips at a temperature setting of 22degrees. The whole cells configuration was formed with default Qube sealand break-in parameter. The membrane potential was held at −100 mVbefore the voltage protocol began. Two voltage protocols were followed.

Step 1: Cells were held at −100 mV with a depolarized pulse to −20 mVfor 10 ms, the interval was set at 5 s. Currents were corrected bydefault leak subtraction from every pulse. The duration was set at 5mins.

Step 2: Cells were held at −100 mV with a depolarized pulse to −20 mVfor 10 ms. The frequency was 5 Hz. Currents were corrected by leaksubtraction calculated before step 2. The duration was set at 4 mins.

Internal Block by Test Compounds

After Step 2, the Qchip was removed from the recording chamber. Theinternal solution was changed with solutions containing test compounds.Qchips were held at −100 mV without pulsing after being replaced in therecording chamber. The total solution switching time was 8 minutes.After the internal solution exchange, Cells were recorded and step 2 forrepeated for 10 minutes.

Data analysis was performed by Sophion Analyzer. Cells were filteredwith minimum 50 MOhm seal resistance and minimum 5 nA starting current.The rundown of the currents was corrected with control cells (Non-Drug).The remaining was calculated by averaging last 3 points in the end ofexperiments. The base line was calculated as the average of last 3points of Step 2. IC₅₀ curves were plotted with DR-plots/Hill function(a dose-response plot with a Hill fit). Data analysis was performed bySophion Analyzer.

Representative examples of the invention were tested for intracellularinhibition of NaV 1.7 in the automated patch clamp assay. Activity Rangeis reported as IC₅₀: “++++” (<1 μM), “+++” (1-3 μM), “++” (3-10 μM) or“+” (10-30 μM). The results are presented in Table M.

TABLE M Nav1.7 Nav1.7 Nav1.7 Com- Intracellular Com- Intracellular Com-Intracellular pound Inhibition pound Inhibition pound Inhibition 6 + + +59 + 88 + + 12 + + + 66 + + + 89 + + 16 + + + 67 + + 90 + + 21 + + + +68 + + + 91 + + 22 + + 69 + 92 + + + 23 + + + + 70 + 93 + + 24 + +71 + + + + 94 + + + + 27 + + + 72 + + 95 + + + 28 + + + + 73 + + +96 + + 29 + + 74 + + 97 + 30 + + + 75 + + + + 98 + + + 31 + + + 76 + +98 + + + 32 + + + 77 + + + + 100 + + + 33 + + + 78 + + + 101 + +34 + + + + 79 + + + 102 + + + 40 + + + 80 + + + 103 + + 46 + + 81 + + +104 + + + 47 + + + 82 + + + 105 + + + 49 + + 83 + + + 106 + + + 50 + + +84 + + 107 + + 51 + + + + 85 + + 108 + + + 53 + 86 + + 109 + + + +54 + + 87 + + + + 110 +

External Block by Test Compounds

After Step 2, the external solution was changed with solutionscontaining test compounds. Qchips were held at −100 mV without pulsing.The total solution switching time was 8 minutes. After the externalsolution exchange, cells were recorded using the same procedure as step2 for 10 minutes.

Data analysis was performed by Sophion Analyzer. Data were correctedwith control cells (Non-Drug). The IC₅₀ data were plotted withDR-plots/Hill function (a dose-response plot with a Hill fit).

Representative examples of the invention were tested for extracellularinhibition of NaV 1.7 in the automated patch clamp assay. Activity Rangeis reported as IC₅₀: “+++” (<10 μM), “+++” (10-30 μM), “++” (30-100 μM)or “+” (>100 μM). The results are presented in Table N.

TABLE N Nav1.7 Nav1.7 Nav1.7 Com- Extracellular Com- Extracellular Com-Extracellular pound Inhibition pound Inhibition pound Inhibition 6 +54 + + 87 + 12 + + + 66 + + 88 + 16 + 67 + + + + 89 + 21 + 68 + 90 +22 + 69 + 91 + + 23 + 70 + 92 + 24 + 71 + 93 + 27 + 72 + 94 + + 28 +73 + 95 + 29 + 74 + 96 + + 30 + 75 + 97 + + + + 31 + 76 + 98 + 32 +77 + + + 100 + + + + 33 + 78 + 101 + 34 + + + + 79 + + 102 + 40 + 80 +103 + + 46 + 81 + 104 + 47 + 82 + 106 + 49 + 83 + 107 + 50 + 84 + 108 +50 + 85 + 109 + 51 + 86 +

Example 4—Membrane Permeability

The PAMPA assay (pION, Inc., Woburn Mass.) was used to determine theability of compounds of the invention to cross an artificial lipidmembrane by passive diffusion. Test compounds were dissolved in DMSO (10mM) and diluted 200-fold in buffer (pION Inc., pH 7.4) to provide 50 uMstock solutions. Buffer (150 μL) was added to a UV blank plate and stocksolutions (150 μL) were transferred to a UV reference plate. The blankand reference spectrum were read using a spectrophotometer. Stocksolutions (200 μL) were added to the donor plate of the PAMPA sandwichplate and an accept plate painted with GIT lipid (pION Inc, 5 μL) wasplaced on top. Buffer (200 μL) was added to the acceptor plate and thePAMPA sandwich plate was incubated for 4 hours. Aliquots (150 μL) fromthe acceptor plate were added to a UV plate and read as acceptorspectrum. Aliquots (150 μL) of the donor solutions were added to a UVanalysis plate and read as donor spectrum. The permeability coefficientof test compounds was calculated using PAMPA Explorer™ software (version3.5.0.4) based on the AUC of the reference plate, the donor plate, andthe acceptor plate.

The PAMPA permeability results (10⁻⁶ cm/s) of representative compoundsare reported as “+” (<0.1 10⁻⁶ cm/s), “++” (0.1-2.0 10⁻⁶ cm/s), “+++”(2.0-10.010⁻⁶ cm/s) or “++++” (>10.0 10⁻⁶ cm/s) (Table O).

TABLE O Com- PAMPA Com- PAMPA Com- PAMPA pound (10⁻⁶cm/s) pound(10⁻⁶cm/s) pound (10⁻⁶cm/s) 1A + + 34 + + 78 + 2A + 36 + 80 + 3A + +41 + 81 + 4A + 46 + 82 + 5A + 47 + 83 + 6A + + 48 + 84 + 7A + 50 + 85 +8A + 51 + + 86 + 9A + 53 + 87 + 10A + 54 + + 88 + 11A + 58 + 89 + 12A +59 + + 90 + 13A + 60 + + + 91 + 14A + 62 + 92 + 15A + + 63 + 93 +16A + + 64 + + 94 + 17A + 66 + + 95 + 18A + 68 + 98 + 21A + 69 + 101 +23A + 70 + 102 + 24A + 71 + 103 + 25A + 72 + 104 + 27A + 73 + 106 +28A + 74 + 107 + 30A + + 75 + 108 + + 31 + + 76 + 109 + + 33 + + 77 +110 + +

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims. It will also be understood that noneof the embodiments described herein are mutually exclusive and may becombined in various ways without departing from the scope of theinvention encompassed by the appended claims.

What is claimed is:
 1. A compound represented by Formula (I)

wherein: Y⁻ is a pharmaceutically acceptable anion; R^(F) and R^(G)together with the N⁺ to which they are attached form an optionallysubstituted heteroaryl ring having zero, one or more heteroatoms inaddition to the N⁺, or an optionally substituted bicyclic heteroarylring having zero, one or more heteroatoms in addition to the N⁺; R^(A),R^(B), and R^(C) are each independently selected from H, D halogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, nitrile, OR^(I),NR^(J)R^(K), NR^(L)C(O)R^(M), S(O)R^(N), SO₂R^(O), SO₂R^(O)R^(P),SO₂NR^(Q)R^(R), SO₃R^(S), CO₂R^(T), C(O)R^(U), and C(O)NR^(V)R^(W); orR^(B) and vicinal R^(C) together with the carbon atoms to which they areattached form a substituted or unsubstituted 3-7-membered cycloalkyl ora substituted or unsubstituted aryl; each of R^(I), R^(J), R^(K), R^(L),R^(M), R^(N), R^(O), R^(P), R^(Q), R^(R), R^(S), R^(T), R^(U), R^(V),and R^(W) is independently selected from H, D, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, orsubstituted or unsubstituted alkynyl; X¹ is selected from —CR^(X)R^(Y)—,—NR^(Z)C(O)—, —NR^(Z)C(O)CR^(X)R^(Y)—, —OC(O)—, —SC(O)—, —C(O)NR^(1A)—,—C(O)O—, —NR^(Z)S(O)—, —S(O)NR^(Z)—, —NR^(X)C(O)NR^(Y)—, —(O)CS—,—C(O)—, —S(O)—, and —S(O)₂—; each of R^(X), R^(Y), R^(Z), and R^(1A) isindependently selected from H, D, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl; and each of R^(D) and R^(E) is independently selected from H,D, substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, cycloalkyl, aryl, orheteroaryl; or R^(D) and R^(E) together with the carbon to which theyare attached form a substituted or unsubstituted C₃-C₇ cycloalkyl or asubstituted or unsubstituted heterocyclic ring; or R^(D) and R^(Z)together with the carbon and the —N—C(O)— to which they are attachedform an optionally substituted 5-8-membered lactam.
 2. The compound ofclaim 1, wherein Y⁻ is iodide, bromide, or chloride.
 3. The compound ofclaim 1, wherein X¹ is —NHC(O)—.
 4. The compound of claim 1, whereineach of R^(A) and R^(B) is independently selected from H, D, nitrile,halogen, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, and NR^(J)R^(K); andeach of R^(J) and R^(K) is independently selected from H, substituted orunsubstituted C₁₋₄ alkyl, substituted or unsubstituted C₂₋₄ alkenyl, andsubstituted or unsubstituted C₂₋₄ alkynyl.
 5. The compound of claim 1,wherein R^(A), R^(B), and R^(C) are each independently selected from H,D, halogen, OR^(I), substituted or unsubstituted C₁-C₄ alkyl, andNR^(J)R^(K); wherein each of R^(I), R^(J) and R^(K) is independentlyselected from H and substituted or unsubstituted C₁-C₄ alkyl.
 6. Thecompound of claim 1, wherein each of R^(A) and R^(B) is CH₃, and R^(C)is selected from the group consisting of H, CH₃, halogen, nitrile,methoxy, and ethoxy.
 7. The compound of claim 1, wherein R^(D) is C₁₋₄alkyl optionally substituted with a substituent selected from the groupconsisting of halogen, oxygen, C₃₋₅ cyclic alkyl, aryl, and heteroaryl.8. The compound of claim 1, wherein R^(E) is H, D, or C₁₋₄ alkyloptionally substituted with a substituent selected from the groupconsisting of halogen, oxygen, C₃₋₈ cyclic alkyl, aryl, and heteroaryl.9. The compound of claim 1, wherein R^(D) and R^(E) are both hydrogen.10. The compound of claim 1, wherein R^(D) is hydrogen and R^(E) is anC₁-C₆ alkyl.
 11. The compound of claim 1, wherein R^(D) and R^(E) aretaken together with the carbon to which they are attached to form aC₃-C₆ cycloalkyl including, but not limited to, cyclopropyl andcyclobutyl.
 12. The compound of claim 1, wherein R^(F) and R^(G)together with the N⁺ form an optionally substituted heteroaryl ring oran optionally substituted bicyclic heteroaryl ring comprising one ormore heteroatoms selected from the following:


13. The compound of claim 1, wherein R^(F) and R^(G) together with theN⁺ form an optionally substituted pyridinium ring.
 14. The compound ofclaim 1, wherein the heteroaryl ring or bicyclic heteroaryl ring formedby R^(F) and R^(G) together is optionally substituted with a substituentselected from the group consisting of substituted or unsubstituted C₁-C₆alkyl, substituted or unsubstituted C₃-C₆ cycloalkyl, substituted andunsubstituted 3- to 15-membered heterocyclyl, alkoxy, or CO₂R^(2A),wherein R^(2A) is selected from H, D, substituted or unsubstitutedalkyl, substituted or unsubstituted alkenyl.
 15. The compound of claim1, wherein the compound is selected from those below: Com- poundChemical Structure Com- pound 1

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16. A pharmaceutical composition comprising the compound of claim 1 or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable excipient.
 17. The composition of claim 16, wherein saidcomposition is formulated for oral, intravenous, intramuscular, rectal,cutaneous, subcutaneous, topical, transdermal, sublingual, nasal,inhalation, vaginal, intrathecal, epidural, or ocular administration.18. A method for treating pain, cough, itch, or a neurogenicinflammatory disorder in a patient, comprising administering to saidpatient an effective of a compound of claim
 1. 19. The method of claim18, wherein said pain is selected from the group consisting of pain dueto back and neck pain, lower back pain, cancer pain, gynecological andlabor pain, fibromyalgia, arthritis, rheumatoid arthritis,osteoarthritis, rheumatological pains, orthopedic pains, acute and postherpetic neuralgia and other neuropathic pains (including peripheralneuropathy), sickle cell crises, vulvodynia, peri-anal pain, irritablebowel disease, irritable bowel syndrome, inflammatory bowel disease,oral mucositis, esophagitis, interstitial cystitis, urethritis and otherurological pains, dental pain, headaches, trigeminal trophic syndrome,erythromelalgia, abdominal wall pain, chronic abdominal wall pain,allergic rhinitis, muscle pain, rectal pain, Levator ani syndrome,proctalgia fugax, hemorrhoid pain, stomach pain, skin ulcers, stomachulcers, burn pain, ophthalmic irritation, conjunctivitis (e.g., allergicconjunctivitis), eye redness, dry eye, dry eye syndrome (chronic ocularpain), complex regional pain syndrome, post-surgical ocular pain,postoperative pain, acute postoperative pain, and procedural pain (i.e.,pain associated with injections, draining an abscess, surgery, dentalprocedures, ophthalmic procedures, arthroscopies and use of othermedical instrumentation, cosmetic surgical procedures, dermatologicalprocedures, setting fractures, biopsies, and the like).
 20. The methodof claim 18, wherein said cough is selected from the group consisting ofcough in patients with asthma, COPD, asthma-COPD overlap syndrome(ACOS), interstitial pulmonary fibrosis (IPF), idiopathic pulmonaryfibrosis, post viral cough, post-infection cough, chronic idiopathiccough and lung cancer.
 21. The method of claim 18, wherein said itch isselected from the group consisting of itch due to pruritus,brachioradial pruritus, chronic idiopathic pruritus, genital/analpruritus, notalgia paresthetica, scalp pruritus, allergic dermatitis,contact dermatitis, atopic dermatitis, hand eczema, poison ivy,infections, parasites, insect bites, pregnancy, metabolic disorders,liver or renal failure, drug reactions, allergic reactions, eczema,genital and anal itch, hemorrhoid itch, and cancer.
 22. The method ofclaim 18, wherein said neurogenic inflammatory disorder is selected fromthe group consisting of allergic inflammation, asthma, chronic cough,conjunctivitis, rhinitis, psoriasis, inflammatory bowel disease,interstitial cystitis, arthritis, colitis, contact dermatitis, diabetes,eczema, cystitis, gastritis, migraine headache, rosacea, sunburn,pancreatitis, chronic rhinosinusistis, traumatic brain injury,polymicrobial sepsis, tendinopathies, chronic urticaria, rheumaticdisease, acute lung injury, exposure to irritants, inhalation ofirritants, pollutants, chemical warfare agents, and atopic dermatitis.23. The method of claim 18, wherein a compound represented by Formula(I) is used in combination with one or more exogenous large porereceptor agonists.